Pseudouridine (ψ) is the most abundant post-transcriptional RNA modification, yet little is known about the prevalence, mechanism, and function of ψ in mRNA.Here, we perform quantitative mass spectrometry analysis and show that ψ is much more prevalent (ψ/U ratio: ～ 0.2-0.6) in mammalian mRNA than previously believed.We develop CeU-Seq—a selective chemical labeling and pull-down method to identify 2084 ψ sites within 1929 human transcripts, of which four (in rRNA and EEF1A1 mRNA) are biochemically verified.