Sleeping sickness is a re-emerging disease in the Serengeti ecosystem affecting both local people and tourists. Here we report the results of a survey to assess the prevalence of trypanosomiasis in both domestic and wild animals from this area. Five hundred and eighteen cattle samples were collected from 12 villages that bordered the Serengeti National Park and 220 samples from 15 different wild animal species were collected from within the park. PCR analysis, directed against the human serum resistance associated gene SRA, identified human infective Trypanosoma brucei rhodesiense parasites in both cattle and warthogs.