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Background Geographic barriers to gene flow and divergence among populations in sexual traits are two important causes of genetic isolation which may lead to speciation. Genetic isolation may be facilitated if these two mechanisms act synergistically. The guppy from the Cumaná region (within the Cariaco drainage) of eastern Venezuela has been previously described as a case of incipient speciation driven by sexual selection, significantly differentiated in sexual colouration and body shape from the common guppy, Poecilia reticulata. The latter occurs widely in northern Venezuela, including the south-eastern side of Cordillera de la Costa, where it inhabits streams belonging to the San Juan drainage. Here, we present molecular and morphological analyses of differentiation among guppy populations in the Cariaco and San Juan drainages. Our analyses are based on a 953 bp long mtDNA fragment, a set of 15 microsatellites (519 fish from 20 populations), and four phenotypic traits. Results Both microsatellite and mtDNA data showed that guppies inhabiting the two drainages are characterised by a significant genetic differentiation, but a higher proportion of the genetic variance was distributed among populations within regions. Most guppies in the Cariaco drainage had mtDNA from a distinct lineage, but we also found evidence for widespread introgression of mtDNA from the San Juan drainage into the Cariaco drainage. Phenotypically, populations in the two regions differed significantly only in the number of black crescents. Phenotypic clustering did not support existence of two distinct groupings, but indicated a degree of distinctiveness of Central Cumaná (CC) population. owever, CC population showed little differentiation at the neutral markers from the proximate populations within the Cariaco drainage. Conclusions Our findings are consistent with only partial genetic isolation between the two geographic regions and indicate that the geographic barrier of Cordillera de la Costa has not played an important role in strengthening the incomplete pre-zygotic reproductive barrier between Cumaná and common guppy. Significant phenotypic differentiation between genetically similar (in terms of neutral variation) populations suggests that mate choice can maintain divergence at sexually selected traits despite gene flow. However, neither genetic nor phenotypic clustering supported delineation of two species within the region. [ABSTRACT FROM AUTHOR]