The article introduces a novel method called Ultrafast Bisulfite Sequencing (UBS-seq), which addresses the limitations of conventional Bisulfite Sequencing (BS-seq) in detecting 5-methylcytosine (5mC) in DNA and RNA. UBS-seq uses highly concentrated bisulfite reagents and higher reaction temperatures to accelerate the bisulfite reaction, reducing DNA damage and background noise. This method allows for library construction from small amounts of genomic DNA, such as cell-free DNA or mouse embryonic stem cells, with less overestimation of 5mC levels and higher genome coverage compared to conventional BS-seq. UBS-seq also enables quantitative mapping of RNA 5mC from low-input mRNA samples and detects m5C stoichiometry in highly structured RNA sequences. The study identifies NSUN2 as the major 'writer' protein responsible for ~90% of m5C sites in HeLa mRNA and reveals enriched m5C sites in 5′-regions of mammalian mRNA, suggesting functional roles in mRNA translation regulation. UBS-seq outperforms conventional BS-seq in terms of reduced background, less 5mC level overestimation, and higher genome coverage, making it a powerful tool for both DNA and RNA 5mC sequencing.The article introduces a novel method called Ultrafast Bisulfite Sequencing (UBS-seq), which addresses the limitations of conventional Bisulfite Sequencing (BS-seq) in detecting 5-methylcytosine (5mC) in DNA and RNA. UBS-seq uses highly concentrated bisulfite reagents and higher reaction temperatures to accelerate the bisulfite reaction, reducing DNA damage and background noise. This method allows for library construction from small amounts of genomic DNA, such as cell-free DNA or mouse embryonic stem cells, with less overestimation of 5mC levels and higher genome coverage compared to conventional BS-seq. UBS-seq also enables quantitative mapping of RNA 5mC from low-input mRNA samples and detects m5C stoichiometry in highly structured RNA sequences. The study identifies NSUN2 as the major 'writer' protein responsible for ~90% of m5C sites in HeLa mRNA and reveals enriched m5C sites in 5′-regions of mammalian mRNA, suggesting functional roles in mRNA translation regulation. UBS-seq outperforms conventional BS-seq in terms of reduced background, less 5mC level overestimation, and higher genome coverage, making it a powerful tool for both DNA and RNA 5mC sequencing.