The trypanosomes include a number of species that cause disease in livestock. In recent years, several trypanosomes have been identified which do not fit into the classic trypanosome classification system. However, previous work has focused on trypanosomes identified in the tsetse vector, with little information available on trypanosomes found in their natural hosts, wildlife. We studied trypanosome sequences from wildlife in Serengeti National Park in Tanzania and the Luangwa Valley in Zambia and found a number of trypanosome species pathogenic to livestock were circulating in these areas. For Trypanosoma vivax, one of the causes of trypanosomiasis in cattle, variants were identified in giraffe and waterbuck that were different from all published sequences and from each other. These variants did not test positive with the molecular tests usually used to identify T. vivax suggesting that T. vivax may often be under-diagnosed in Tanzania. The trypanosome classification system is facing challenges as molecular data are incorporated into a system that historically was based on factors such as morphology, host range and geographical distribution.