Cyclotides are a novel family of plant-derived defense peptides that are biosynthetically produced via the processing of cyclotide precursor (CP) proteins containing one, two or three cyclotide domains. By screening a cDNA library of Viola baoshanensis roots and using RACE and RT-PCR methods, 23 cDNA clones were identified and then used to deduce full CP proteins containing one (VbCP1S-5), two (VbCP6S), or three (VbCP7S) cyclotide domains. RT-PCR and sequence analyses suggested that VbCP6S were resulted from the alternative splicing of VbCP7S RNA. The significance of VbCP7S RNA splicing is that it provides a mechanism for increasing the diversity of cyclotide expression via the recombination of N-terminal repeat (NTR) regions and cyclotide domains. After analyzing the full endoplasmic reticulum (ER) signals of known and novel CPs associated with RT-PCR tests, three primers encoding the conserved sequence ALVLIATFA, AAFALPA-LA and AAFALPA-AFA were proposed to be more efficient in cloning CP genes than the well-applied primer encoding AAFALPA. Cyclotide sequence analyses indicated that the cDNA clones encoded a variety of Möbius and bracelet cyclotides, which were likely involved in the known bioactivities of cyclotides, and also might play a previously unreported role in mediating the metal tolerance of V. baoshanensis. Overall, this study shows that CP genes are varied in V. baoshanensis and cyclotide expression is subject to transcriptional and post-transcriptional regulation in this plant.