Post-mating reproductive isolating mechanisms may be among the earliest reproductive barriers to emerge among incipient species. Trinidadian guppy, Poecilia reticulata , populations in the Caroni and Oropouche drainages in Northern Trinidad exhibit marked genetic divergence and provide an ideal system in which to search for these barriers. We inseminated virgin females with equal amounts of sperm from two males, a ‘native’ male from the female's own population and a ‘foreign’ male from the other drainage. Artificial insemination ensured that mating order and mate choice did not affect the outcome. Paternities were assigned to the resulting broods using microsatellite markers. As predicted, sperm from native males had precedence over foreign sperm. Moreover, this effect was symmetrical for both drainages. In contrast, we detected no native sperm precedence in controls, in which females received sperm from the same and another population within the same drainage. Our results show that gametic isolation can arise between geographically proximate, though genetically divergent, populations of a single species and highlight the potential role of this process in speciation.