Abstract5‐Formylcytosine (f⁵C) modification is present in human mitochondrial methionine tRNA (mt‐tRNA^Met) and cytosolic leucine tRNA (ct‐tRNA^Leu), with their formation mediated by NSUN3 and ALKBH1. f⁵C has also been detected in yeast mRNA and human tRNA, but its transcriptome‐wide distribution in mammals has not been studied. Here we report f⁵C‐seq, a quantitative sequencing method to map f⁵C transcriptome‐wide in HeLa and mouse embryonic stem cells (mESCs). We show that f⁵C in RNA can be reduced to dihydrouracil (DHU) by pic‐borane, and DHU can be exclusively read as T during reverse transcription (RT) reaction, allowing the detection and quantification of f⁵C sites by a unique C‐to‐T mutation signature. We validated f⁵C‐seq by identifying and quantifying the two known f⁵C sites in tRNA, in which the f⁵C modification fractions dropped significantly in ALKBH1‐depleted cells. By applying f⁵C‐seq to chromatin‐associated RNA (caRNA), we identified several highly modified f⁵C sites in HeLa and mouse embryonic stem cells (mESC).