Accurate and systematic transcriptome-wide detection of 5-methylcytosine (m5C) has proved challenging, and there are conflicting views about the prevalence of this modification in mRNAs. Here we report an experimental and computational framework that robustly identified mRNA m5C sites and determined sequence motifs and structural features associated with the modification using a set of high-confidence sites. We developed a quantitative atlas of RNA m5C sites in human and mouse tissues based on our framework. In a given tissue, we typically identified several hundred exonic m5C sites. About 62-70% of the sites had low methylation levels (<20% methylation), while 8-10% of the sites were moderately or highly methylated (>40% methylation). Cross-species analysis revealed that species, rather than tissue type, was the primary determinant of methylation levels, indicating strong cis-directed regulation of RNA methylation. Combined, these data provide a valuable resource for identifying the regulation and functions of RNA methylation.