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Abstract The viviparity-driven conflict hypothesis postulates that the evolution of matrotrophy (postfertilization maternal provisioning) will result in a shift from a pre- to postcopulatory mate choice and thus accelerate the evolution of postcopulatory reproductive isolation. Here, we perform artificial insemination experiments on Heterandria formosa, a matrotrophic poeciliid fish, to probe for evidence of postcopulatory female choice. We established laboratory populations from Wacissa River (WR) and Lake Jackson (LJ). The WR females normally produce larger offspring than the LJ females. We artificially inseminated females with sperm from each population or from both populations simultaneously. When LJ females were inseminated with sperm from WR and LJ males, they allocated fewer resources to WR-sired offspring than when they were inseminated with WR sperm alone. The LJ females carrying developing offspring sired by males from different populations were thus able to discriminate against non-resident males when allocating resources to developing young. The WR females, which normally produce larger offspring than LJ females, did not discriminate among males from different localities. These findings provide insights into the ability of females from one population to exercise a form of postcopulatory mate selection.