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African animal trypanosomosis (AAT), is an infectious parasitic disease of wildlife and livestock caused by multiple species and strains of <i>Trypanosoma</i>. In South Africa, it is restricted to northern KwaZulu-Natal (NKZN) and caused by <i>Trypanosoma congolense</i> and <i>Trypanosoma vivax</i>. A cross-sectional study was done to determine AAT prevalence in 384 goat samples and identify trypanosome species circulating in 60 cattle at dip tanks that are on the interface with the Hluhluwe-uMfolozi game reserve in NKZN. Both cattle and goat samples were analyzed using the buffy coat technique (BCT) and a polymerase chain reaction (PCR) assay targeting the internal transcribed spacer 1 (ITS) region. Cattle samples were further analyzed using an ITS quantitative real-time PCR (qPCR) assays designed for the detection of <i>T. congolense</i>, <i>T. vivax,</i> and <i>T. brucei</i>. None of the goat samples tested positive for <i>Trypanosoma</i> infections. The ITS qPCR assay detected <i>Trypanosoma</i> DNA in 30% of the cattle samples, while only 8.3% were positive with the ITS PCR and 11.7% were positive using BCT. Quantitative real-time PCR assays were designed to amplify a 98 bp, 137 bp, and 116 bp fragment of the cathepsin L-like (CATL) gene from <i>T. brucei</i>, <i>T. theileri,</i> and <i>T. congolense</i>, respectively. Each assay was shown to be efficient (>94%) and specific (10⁹ to 10²/10¹ copies/reaction) in the detection of <i>Trypanosoma</i> species. The CATL qPCR assays detected <i>T. congolense</i> and <i>T. theileri</i> infections in 33.3% of the cattle samples. The CATL qPCR assays also detected <i>T. congolense</i> infections in goats (23.1%) that were neither detected by BCT nor the ITS PCR. The CATL qPCR assays provide an additional, sensitive, and specific tool for <i>Trypanosoma</i> diagnostics. The presence of trypanosomes in goats suggests they might be potential reservoirs of infections to other livestock.