AbstractPseudouridine (Ψ) is the most abundant post‐transcriptional RNA modification. Methods have been developed for locus‐specific Ψ detection; however, they often involve radiolabeling of RNA, require advanced experimental skills, and can be time‐consuming. Herein we report a radiolabeling‐free, qPCR‐based method to rapidly detect locus‐specific Ψ. Pseudouridine residues were labeled chemically, and the resulting adducts induced mutation/deletion during reverse transcription (RT) to generate qPCR products with different melting temperatures and hence altered melting curves. We validated our method on known Ψ sites in rRNA and then used it to sensitively detect Ψ residues in lncRNA and mRNA of low abundance. Finally, we applied our method to pseudouridine synthase identification and showed that Ψ616 in PSME2 mRNA is dependent on PUS7. Our facile and cost‐effective method takes only 1.5 days to complete, and with slight adjustment it can be applied to the detection of other epitranscriptomic marks.