The northern KwaZulu-Natal (NKZN) region of South Africa is the southern limit of the African tsetse belt. Entomological information on Glossina brevipalpis and Glossina austeni was generated following the outbreak of trypanosomosis in cattle in 1990. However, these data have not been supported by parallel studies on epidemiology of the disease and therefore there has been no control policy in place. This study presented the first intensive investigations to address the epidemiology of trypanosomosis in NKZN. Tsetse abundance, trypanosome herd average prevalence (HAP), herd average anaemia (HAA) and herd average packed cell volume (HA-PCV) were investigated at three communal diptanks located at the edge of Hluhluwe-iMfolozi Park by monthly sampling from June 2006 – November 2007. Seasonal trypanosome surveys were conducted at seven other communal diptanks. Glossina brevipalpis prevalence was high at two of the diptanks, Mvutshini and Ekuphindisweni, but low at Ocilwane, whilst G. austeni was only collected from Mvutshini. This high and low tsetse challenge presented different disease scenarios. Cattle at Mvutshini and Ekuphindisweni had the highest HAP of 12.3% and 8.9% respectively, both significantly different (p = 0.001) from the HAP obtained from cattle at Ocilwane (2.9%). These two cattle herds also had the highest HAA, 27.7% and 33.4% respectively, whilst cattle at Ocilwane had the lowest, 11.1% (p = 0.001). Conversely, cattle at Ocilwane had the highest HA-PCV, ranging between 29.0% and 32.0%, whilst cattle at Mvutshini and Ekuphindisweni had the lowest HA-PCV (24.0% – 29.0%). By combining the data from the three diptanks (1318 observations), 62.0% of the infected cattle were found anaemic, compared to 20.0% in the uninfected group. Trypanosome seasonal surveys showed that cattle at all the seven diptanks were infected with trypanosomes; mean HAP, HAA and HA-PCV of 10.2%, 46.6% and 23.7%, respectively. This study generated information on the epidemiological factors related to the wide spread of trypanosome-infected cattle and tsetse flies. Trypanosomosis is a disease of economic importance impacting the livelihood of resource-poor farmers in NKZN.