Three-spined sticklebacks (Gasterosteus aculeatus) are a powerful evolutionary model system due to the rapid and repeated phenotypic divergence of freshwater forms from a marine ancestor throughout the Northern Hemisphere. Many of these recently derived populations are found in overlapping habitats, yet are reproductively isolated from each other. This scenario provides excellent opportunities to investigate the mechanisms driving speciation in natural populations. Genetically distinguishing between such recently derived species, however, can create difficulties in exploring the ecological and genetic factors defining species boundaries, an essential component to our understanding of speciation. We overcame these limitations and increased the power of analyses by selecting highly discriminatory markers from the battery of genetic markers now available. Using species diagnostic molecular profiles, we quantified levels of hybridization and introgression within three sympatric species pairs of three-spined stickleback. Sticklebacks within Priest and Paxton lakes exhibit a low level of natural hybridization and provide support for the role of reinforcement in maintaining distinct species in sympatry. In contrast, our study provides further evidence for a continued breakdown of the Enos Lake species pair into a hybrid swarm, with biased introgression of the ‘limnetic’ species into that of the ‘benthic’; a situation that highlights the delicate balance between persistence and breakdown of reproductive barriers between young species. A similar strategy utilizing the stickleback microsatellite resource can also be applied to answer an array of biological questions in other species’ pair systems in this geographically widespread and phenotypically diverse model organism.