In total, 67 novel diamidine compounds were tested in vitro to determine activity against an animal pathogenic Chinese kinetoplastic T. evansi strain. In comparison, a human pathogenic Trypanosoma brucei rhodesiense strain and a P2 transporter knock out of aTrypanosoma brucei brucei strain were also tested. All diamidine compounds tested in this study against T. evansi produced inhibitory concentration (IC50) values below 50 nM. The results demonstrate that these compounds are highly active against T. evansi in vitro. In addition, preliminary in vivo toxicity tests were performed on all 67 diamidines with 69 % of the compounds showing no acute toxicity at an intra-peritoneal dose of 100 mg/kg.