RNA molecules are modified post-transcriptionally to acquire diverse functions. Transfer RNA (tRNA) has the most extensive and numerous RNA modifications. These modifications are crucial for decoding the genetic code and stabilizing tRNA structure. Alterations in tRNA modifications directly affect tRNA structure and function, regulating gene expression. Thermophilic organisms exhibit unique tRNA modifications that dynamically respond to temperature changes, enhancing their thermotolerance. This review summarizes the history and recent findings on tRNA modifications that contribute to thermotolerance in thermophiles. tRNA modifications include methylation, hydroxylation, acetylation, deamination, isomerization, selenylation, reduction, cyclization, and conjugation with amino acids and sugars. These modifications stabilize RNA structures, contribute to various biological functions, and influence RNA interactions with proteins, processing, and translation. RNA modifications also act as molecular zip codes for intracellular localization and help evade the innate immune system. RNA modifications are linked to human diseases, including cancer, diabetes, and neuromuscular disorders. Dysregulation of RNA modifications can lead to disease, and drugs targeting RNA-modifying enzymes are being developed. tRNA contains the most diverse and numerous RNA modifications, with approximately 80% of known RNA modifications being tRNA modifications. These modifications are clustered in the anticodon and core regions of tRNA, playing critical roles in protein synthesis and tRNA stability. Thermophilic organisms have unique tRNA modifications that enhance their thermal stability and resistance to degradation. These modifications include 2'-O-methylation, pseudouridine, 5-methyluridine, 4-thiouridine, and 7-methylguanosine. These modifications stabilize tRNA structure, enhance thermal stability, and contribute to efficient translation. For example, 2'-O-methylation stabilizes the tRNA structure by taking C3'-endo ribose puckering, while pseudouridine forms hydrogen bonds to stabilize the tRNA structure. Thermophilic tRNA modifications are dynamically regulated in response to temperature changes. For instance, the modification m⁵s²U54 is highly conserved and contributes to thermal stability. The frequency of this modification increases with growth temperature, enhancing tRNA stability and protein synthesis. Similarly, m⁷G46 is a prevalent modification in various RNA species, contributing to RNA stability and thermotolerance. Other tRNA modifications, such as ac⁴C, also play critical roles in RNA stability and thermotolerance. ac⁴C is found in various RNA species and contributes to RNA duplex stability. In thermophilic archaea, ac⁴C is abundant and helps maintain RNA structure at high temperatures. The biogenesis of ac⁴C is regulated by specific enzymes, and its presence is essential for RNA stability and thermotolerance. The reversible phosphorylation of tRNA by the Arkl writer and KptA eraser is another important modificationRNA molecules are modified post-transcriptionally to acquire diverse functions. Transfer RNA (tRNA) has the most extensive and numerous RNA modifications. These modifications are crucial for decoding the genetic code and stabilizing tRNA structure. Alterations in tRNA modifications directly affect tRNA structure and function, regulating gene expression. Thermophilic organisms exhibit unique tRNA modifications that dynamically respond to temperature changes, enhancing their thermotolerance. This review summarizes the history and recent findings on tRNA modifications that contribute to thermotolerance in thermophiles. tRNA modifications include methylation, hydroxylation, acetylation, deamination, isomerization, selenylation, reduction, cyclization, and conjugation with amino acids and sugars. These modifications stabilize RNA structures, contribute to various biological functions, and influence RNA interactions with proteins, processing, and translation. RNA modifications also act as molecular zip codes for intracellular localization and help evade the innate immune system. RNA modifications are linked to human diseases, including cancer, diabetes, and neuromuscular disorders. Dysregulation of RNA modifications can lead to disease, and drugs targeting RNA-modifying enzymes are being developed. tRNA contains the most diverse and numerous RNA modifications, with approximately 80% of known RNA modifications being tRNA modifications. These modifications are clustered in the anticodon and core regions of tRNA, playing critical roles in protein synthesis and tRNA stability. Thermophilic organisms have unique tRNA modifications that enhance their thermal stability and resistance to degradation. These modifications include 2'-O-methylation, pseudouridine, 5-methyluridine, 4-thiouridine, and 7-methylguanosine. These modifications stabilize tRNA structure, enhance thermal stability, and contribute to efficient translation. For example, 2'-O-methylation stabilizes the tRNA structure by taking C3'-endo ribose puckering, while pseudouridine forms hydrogen bonds to stabilize the tRNA structure. Thermophilic tRNA modifications are dynamically regulated in response to temperature changes. For instance, the modification m⁵s²U54 is highly conserved and contributes to thermal stability. The frequency of this modification increases with growth temperature, enhancing tRNA stability and protein synthesis. Similarly, m⁷G46 is a prevalent modification in various RNA species, contributing to RNA stability and thermotolerance. Other tRNA modifications, such as ac⁴C, also play critical roles in RNA stability and thermotolerance. ac⁴C is found in various RNA species and contributes to RNA duplex stability. In thermophilic archaea, ac⁴C is abundant and helps maintain RNA structure at high temperatures. The biogenesis of ac⁴C is regulated by specific enzymes, and its presence is essential for RNA stability and thermotolerance. The reversible phosphorylation of tRNA by the Arkl writer and KptA eraser is another important modification