Understanding how reproductive barriers evolve and which barriers contribute to speciation requires the examination of organismal lineages that are still in the process of diversification and the study of the full range of reproductive barriers acting at different life stages. Lake and river ecotypes of the East African cichlid fish Astatotilapia burtoni show habitat specific adaptations, despite different levels of genetic differentiation, and thus represent an ideal model to study the evolution of reproductive barriers. To evaluate the degree of reproductive isolation between genetically divergent lake and river populations, we performed a mesocosm mating experiment in a semi-natural setting at Lake Tanganyika. We assessed reproductive isolation in the presence of male–male competition by analysing survival and growth rates of introduced adults and their reproductive success from genetic parentage of surviving offspring. The genetically divergent river population showed reduced fitness in terms of survival, growth rate, and mating success in a lake-like environment. Hybrid offspring between different populations showed intermediate survival consistent with extrinsic postzygotic reproductive barriers. Our results suggest that both prezygotic (immigrant inviability) and postzygotic reproductive barriers contribute to divergence, and highlight the value of assessing multiple reproductive barriers acting at different stages and in natural contexts to understand speciation mechanisms.