5-Hydroxymethylcytidine (hm(5)C) was recently identified as a direct metabolite of m(5)C in RNA. We investigated the stability of hm(5)C in human cells using bio-isotopologues and LC-MS/HRMS. This has led to the discovery of a second oxidative metabolite of m(5)C in RNA, namely 2'-O-methyl-S-hydroxymethylcytidine (hm(5)Cm). Subsequent quantitative analysis of total RNA from higher organisms revealed varying levels and TET-independent formation of this new RNA modification.