As part of ongoing explorations of the structural diversity of cyclotides, the cyclotide content of a native violet of the East African highlands, Viola abyssinica (which grows at altitudes up to 3400 m), was studied. Six new cyclotides, vaby A-E (1-5) and varv E (6), were isolated and characterized by employing HPLC and MS techniques and quantitative amino acid analysis. Cyclotides 1-5 were found to have new sequences, and 1-3 have a further novel feature in their sequences, an alanine moiety in loop 2. Two of the cyclotides (1 and 4) also exhibited cytotoxic properties in a flourometric microculture cytotoxicity assay. The findings corroborate the hypothesis that investigating the cyclotide contents of violets growing in diverse environments is a promising approach for extending our knowledge of both the structural and biological diversity of cyclotides.