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Museum records indicate that Hybognathus placitus was introduced into the Pecos River, New Mexico during the early 1960s. Approximately 10 years later, a congener, Hybognathus amarus, was extirpated from the system. We used microsatellite and mtDNA data, ecological data and modelling, and a computer simulation approach to reconstruct the history of invasion and species replacement. To identify the potential role of hybridization and introgression, we genetically screened H. amarus (n = 389) from the Rio Grande, New Mexico, and H. placitus (n = 424) from the Pecos River, New Mexico using four nuclear microsatellites and a partial fragment of the mtDNA ND4 gene. Assignment tests excluded hybridization as a primary factor in species replacement and suggested a role for interspecific competition. Genetic analyses showed that H. placitus were introduced into the Pecos River from at least two genetically distinct source populations in the Canadian and Red rivers, Oklahoma. Lotka-Volterra models of interspecific competition indicated that the number of founding individuals could have been as few as 20 for H. placitus to have competitively displaced H. amarus in the Pecos River in 10 to 15 generations. Observed differences of allele frequencies between source and founder populations indicated that between 32 and 115 H. placitus individuals founded the Pecos River. Genetic and ecological data suggest that interspecific competition could have led to species replacement in this arid-land river system.