2'-O-methylation is present within various cellular RNAs and is essential to RNA biogenesis and functionality. Several methods have been developed for the identification and localization of 2'-O-methylated sites in RNAs; however, the detection of RNA modifications, especially in low-abundance RNAs and small non-coding RNAs with a 2'-O-methylation at the 3'-end, remains a difficult task. Here, we introduce a new method to detect 2'-O-methylated sites in diverse RNA species, referred to as RTL-P [Reverse Transcription at Low deoxy-ribonucleoside triphosphate (dNTP) concentrations followed by polymerase chain reaction (PCR)] that demonstrates precise mapping and superior sensitivity compared with previous techniques. The main procedures of RTL-P include a site-specific primer extension by reverse transcriptase at a low dNTP concentration and a semi-quantitative PCR amplification step. No radiolabeled or fluorescent primers are required. By designing specific RT primers, we used RTL-P to detect both previously identified and novel 2'-O-methylated sites in human and yeast ribosomal RNAs (rRNAs), as well as mouse piwi-interacting RNAs (piRNAs). These results demonstrate the powerful application of RTL-P for the systematic analysis of fully or partially methylated residues in diverse RNA species, including low-abundance RNAs or small non-coding RNAs such as piRNAs and microRNAs (miRNAs).