Cyclotides are cyclic cystine knotted macrocyclic plant peptides that have several promising applications. This study was undertaken to detect and identify known and new cyclotides in Viola odorata, a commercially important medicinal plant, from three geographical locations in India. The number of cyclotides in the plant varied with the tissue (leaves, petioles, flowers, runners, and roots) and with geographical locations in India. Using liquid chromatography coupled to Fourier transform mass spectrometry (FTMS), 166 cyclotide-like masses were observed to display cyclotide-diagnostic mass shifts following reduction, alkylation, and digestion, and 71 of these were positively identified based on automated spectrum matching. Of the remaining 95 putative cyclotides observed, de novo peptide sequencing of three new cyclotides, namely, vodo I1 (1), vodo I2 (2), and vodo I3 (3), was carried out with tandem mass spectrometry. © 2017 The American Chemical Society and American Society of Pharmacognosy.