The alien invasive Gambusia affinis is one of the most widely introduced fish species on the planet, and has established in freshwater ecosystems across South Africa. The invasion ecology and, in particular, the population dynamics of the species in this country are, however, poorly understood. In this study the relative abundance and population dynamics of G. affinis were quantified in 5 interconnected irrigation impoundments within the Sundays River Valley, Eastern Cape. Four fish surveys were conducted from early summer (February 2012) to early winter (June 2012). Repeated-measures ANOVA analyses on the catch per unit effort (CPUE) of G. affinis between sampling events and dams revealed significant differences in population dynamics among dams, although an overall trend of rapid increase followed by plateau in summer, with a rapid decline in winter was seen in most dams. A general linear model assessing the role of biotic and abiotic factors on G. affinis CPUE found that water temperature and the presence of the native fish Glossogobius callidus had significant effects on the CPUE of G. affinis. While winter drops in temperature are likely to have caused mortality in G. affinis populations, and may act as the primary regulator of G. affinis establishment success in South African impoundments, the negative effect of G. callidus densities on G. affinis suggests competitive or predator-prey interactions with the native species.