Mapping the position and quantifying the level of 5-methylcytosine (m 5 C) as a modification in different types of cellular RNA is an important objective in the emerging field of epitranscriptomics. Bisulfite conversion has long been the gold standard for detection of m 5 C in DNA but it can also be applied to RNA. Here, we detail methods for bisulfite treatment of RNA, locus-specific PCR amplification and detection of candidate sites by sequencing on the Illumina MiSeq platform.