Little is known about the freshwater fishes of western Ecuador despite serious environmental threats, including the creation of large artificial impoundments. Phenotypic and genetic divergence of populations of a large predatory fish, Hoplias microlepis, is examined in rivers and artificial impoundments of the Guayas River drainage in western Ecuador. Despite the recent formation of the impoundments (∼20 years prior to the sampling), H. microlepis in these habitats diverged morphologically from river populations. Impoundment fish tended to have larger eyes, longer dorsal and caudal fins, and thinner bodies than river fish. Classification rates for habitat of origin based on morphometric measures were relatively high (71.7–83.3%), and the magnitude of morphological divergence was substantial when contrasted with divergence from H. malabaricus, a congener from eastern Ecuador. Frequencies of mtDNA d-loop haplotypes differed significantly among samples. Genetic divergence between river samples implies that the genetic structure in the drainage predates the formation of the impoundments. The genetic analysis also indicates that the morphological divergence between fish in different habitat types is not likely due to shared ancestry. Genetic diversity tended to be higher in the river samples, and the percentage of private alleles was higher in the impoundment populations, which is consistent with rapid population expansion from a limited number of founders in impoundments.