Bisulfite sequencing (BS-seq) to detect 5-methylcytosine (5mC) is limited by lengthy reaction times, severe DNA damage, overestimation of 5mC level and incomplete C-to-U conversion of certain DNA sequences. We present ultrafast BS-seq (UBS-seq), which uses highly concentrated bisulfite reagents and high reaction temperatures to accelerate the bisulfite reaction by ~13-fold, resulting in reduced DNA damage and lower background noise. UBS-seq allows library construction from small amounts of purified genomic DNA, such as from cell-free DNA or directly from 1 to 100 mouse embryonic stem cells, with less overestimation of 5mC level and higher genome coverage than conventional BS-seq. Additionally, UBS-seq quantitatively maps RNA 5-methylcytosine (m⁵C) from low inputs of mRNA and allows the detection of m⁵C stoichiometry in highly structured RNA sequences. Our UBS-seq results identify NSUN2 as the major ‘writer’ protein responsible for the deposition of ~90% of m⁵C sites in HeLa mRNA and reveal enriched m⁵C sites in 5′-regions of mammalian mRNA, which may have functional roles in mRNA translation regulation.