5-Formylcytidine (f5C) is one type of post-transcriptional RNA modification, which is known at the wobble position of tRNA in mitochondria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil, thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. More than 1000 f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA.