A novel method, Nm-REP-seq, was developed for the transcriptome-wide identification of 2-O-methylation sites at single-base resolution by using RNA exoribonuclease (Mycoplasma genitalium RNase R, MgR) and periodate oxidation reactivity to eliminate 2-hydroxylated (2-OH) nucleosides. Nm-REP-seq discovered telomerase RNA component (TERC) RNA, scaRNAs and snoRNAs as new classes of Nm-containing ncRNAs as well as identified many 2-O-methylation sites in various ncRNAs and mRNAs. Furthermore, Nm-REP-seq revealed 2'-O-Methylation located at the 3’-end of snoRNAs, snRNAs, tRNAs and fragments derived from them, as well as piRNAs and miRNAs.