Trypanosoma congolense and Trypanosoma vivax are major species that infect cattle in north-eastern KwaZulu-Natal (KZN), South Africa. Of the two genetically distinct types of T. congolense, Savannah and Kilifi sub-groups, isolated from cattle and tsetse flies in KZN, the former is more prevalent and thought to be responsible for African animal trypanosomosis outbreaks in cattle. Furthermore, variation in pathogenicity within the Savannah sub-group is ascribed to strain differences and seems to be related to geographical locations. The objective of the present study was to compare the virulence of T. congolense strains isolated from African buffaloes (Syncerus caffer) inside Hluhluwe-iMfolozi Park, and from cattle on farms near wildlife parks (< 5 km), to isolates from cattle kept away (> 10 km) from parks. To obtain T. congolense isolates, blood of known parasitologically positive cattle or cattle symptomatically suspect with trypanosomosis, as well as isolates from buffaloes kept inside Hluhluwe-iMfolozi Park were passaged in inbred BALB/c mice. A total of 26 T. congolense isolates were obtained: 5 from buffaloes, 13 from cattle kept near parks and 8 from cattle distant from parks. Molecular characterisation revealed 80% and 20% of isolates to belong to T. congolense Savannah and Kilifi, respectively. To compare virulence, each isolate was inoculated into a group of six mice. No statistical differences were observed in the mean pre-patent period, maximum parasitaemia or drop in packed cell volume (PCV). Significant differences were found in days after infection for the drop in PCV, the patent period and the survival time. These differences were used to categorise the isolates as being of high, moderate or low virulence. Based on the virulence, 12 of 26 (46%) isolates were classified as highly virulent and 27% each as either of moderate or of low virulence. Whilst 11 of 12 high virulent strains were from buffaloes or cattle near the park, only 1 of 7 low virulent strains was from these animals. All the Kilifi T. congolense types were less virulent than the Savannah types. These results confirmed the higher virulence of T. congolense Savannah type compared to Kilifi type and indicated the prevalence of highly virulent strains to be higher in wildlife parks and in cattle near the parks than on farms further away. The geographical location of these strains in relation to the wildlife parks in the area was discussed.