Ultrafast bisulfite sequencing (UBS-seq) is a novel method for detecting 5-methylcytosine (5mC) in DNA and RNA, offering significantly faster reaction times and reduced DNA damage compared to conventional bisulfite sequencing (BS-seq). UBS-seq uses high concentrations of bisulfite reagents and elevated temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in lower background noise and reduced DNA damage. This method allows library construction from small amounts of purified genomic DNA, including cell-free DNA or single cells, with less overestimation of 5mC levels and higher genome coverage than conventional BS-seq. UBS-seq also enables quantitative mapping of RNA 5-methylcytosine (m5C) from low-input mRNA, allowing detection of m5C stoichiometry in highly structured RNA sequences. The study identifies NSUN2 as the major 'writer' protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveals enriched m5C sites in 5'-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation. UBS-seq significantly reduces background noise, minimizing false positives, and allows the detection of thousands of m5C sites in HeLa mRNA, with ~90% of them sensitive to NSUN2 depletion. UBS-seq also shows lower background and less DNA degradation compared to conventional BS-seq, making it suitable for low-input DNA samples and cell-free DNA (cfDNA). The method is particularly effective for detecting m5C in highly structured RNA, such as tRNA, and has been validated for its ability to detect m5C sites in human tRNA with high accuracy and low false positives. UBS-seq was optimized for RNA m5C sequencing, showing efficient C-to-U conversion in RNA without causing severe RNA degradation. It was applied to human mRNA, where it detected a large number of m5C sites with high confidence, showing significant overlap between replicates and good agreement with previous studies. The method also revealed that m5C sites are enriched in 5'-UTR regions of mammalian mRNA, which may play a role 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 5mC sequencing in DNA and RNA. The study highlights the potential of UBS-seq for applications in cancer diagnosis, monitoring, and prognosis, as well as for understanding the biological functions of m5C in RNA.Ultrafast bisulfite sequencing (UBS-seq) is a novel method for detecting 5-methylcytosine (5mC) in DNA and RNA, offering significantly faster reaction times and reduced DNA damage compared to conventional bisulfite sequencing (BS-seq). UBS-seq uses high concentrations of bisulfite reagents and elevated temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in lower background noise and reduced DNA damage. This method allows library construction from small amounts of purified genomic DNA, including cell-free DNA or single cells, with less overestimation of 5mC levels and higher genome coverage than conventional BS-seq. UBS-seq also enables quantitative mapping of RNA 5-methylcytosine (m5C) from low-input mRNA, allowing detection of m5C stoichiometry in highly structured RNA sequences. The study identifies NSUN2 as the major 'writer' protein responsible for the deposition of ~90% of m5C sites in HeLa mRNA and reveals enriched m5C sites in 5'-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation. UBS-seq significantly reduces background noise, minimizing false positives, and allows the detection of thousands of m5C sites in HeLa mRNA, with ~90% of them sensitive to NSUN2 depletion. UBS-seq also shows lower background and less DNA degradation compared to conventional BS-seq, making it suitable for low-input DNA samples and cell-free DNA (cfDNA). The method is particularly effective for detecting m5C in highly structured RNA, such as tRNA, and has been validated for its ability to detect m5C sites in human tRNA with high accuracy and low false positives. UBS-seq was optimized for RNA m5C sequencing, showing efficient C-to-U conversion in RNA without causing severe RNA degradation. It was applied to human mRNA, where it detected a large number of m5C sites with high confidence, showing significant overlap between replicates and good agreement with previous studies. The method also revealed that m5C sites are enriched in 5'-UTR regions of mammalian mRNA, which may play a role 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 5mC sequencing in DNA and RNA. The study highlights the potential of UBS-seq for applications in cancer diagnosis, monitoring, and prognosis, as well as for understanding the biological functions of m5C in RNA.