Small RNAs have the potential to store a secondary layer of labile biological information in the form of modified nucleotides. Emerging evidence has shown that small RNAs including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and tRNA-derived small RNAs (tsRNAs) harbor a diversity of RNA modifications. These findings highlight the importance of RNA modifications in the modulation of basic properties such as RNA stability and other complex physiological processes involved in stress responses, metabolism, immunity, and epigenetic inheritance of environmentally acquired traits, among others. High-resolution, high-throughput methods for detecting, mapping and screening these small RNA modifications now provide opportunities to uncover their diagnostic potential as sensitive disease markers.