The effects of DDT and its metabolites on the reproductive health of two fish species (Clarias gariepinus and Oreochromis mossambicus) were studied using the gonadosomatic index (GSI), gonadal histology and computer assisted sperm analysis (CASA). DDT and its metabolites, DDE and DDD are endocrine disrupting chemicals posing estrogenic and anti-androgenic properties, which have detrimental effects on growth and reproduction. Although DDT was banned internationally, it is still used for Malaria vector control in areas of South Africa. Both species were sampled at a reference site, Albasini dam (AD), (outside the DDT-sprayed area, <0.01 μg L−1 of DDT) and at an exposed site, Xikundu weir (XW), in the same river ± 70 km within the DDT-sprayed area, <0.01 μg L−1 of DDT. Gill nets were used to acquire a sample size of 10 male fish per site for each species. Both testes were dissected out and the GSI calculated. Uncontaminated milt was collected and analyzed using a novel CASA system based on open source software for characterization of sperm motility parameters. Testis tissue sampled for histology was fixed in Bouins and processed according to standard methods. The sample size obtained at the reference site (AD) was O. mossambicus n = 6, C. gariepinus n = 9; and at the exposed site (XW) was O. mossambicus n = 10, C. gariepinus n = 3. The GSI results showed a lower value for O. mossambicus at XW (0.06) (n = 13) than AD (0.12) (n = 6); U = 16.0, P = 0.044. However the opposite was true for C. gariepinus. Exposure to various environmental toxicants can result in gonadal changes such as decreased GSI, morphological alterations or both. Histological assessment showed histopathological alterations to testes tissue including intersex (only in O. mossambicus) and detachment of basal membranes. The CASA results showed a decrease in parameters from the reference site (AD) to the exposed site (XW) for both species. The decrease in percent motility (% MOT) for O. mossambicus from the reference site (AD) (n = 6) to the exposed site (XW) (n = 13) was statistically significant; U = 13.0, P = 0.023. C. gariepinus from the exposed site (XW) was most affected in terms of percent motility (10.00%) and velocity (67.94 μm s−1). Detailed assessment of the environmental effects of DDT in this area seems crucial before population impacts become evident.