The tomato clade within the genus Solanum has numerous advantages for mechanistic studies of reproductive isolation. Its thirteen closely related species, along with four closely allied Solanum species, provide a defined group with diverse mating systems that display complex interspecific reproductive barriers. Several kinds of pre- and postzygotic barriers have already been identified within this clade. Well-developed genetic maps, introgression lines, interspecific bridging lines, and the newly available draft genome sequence of the domesticated tomato (Solanum lycopersicum) are valuable tools for the genetic analysis of interspecific reproductive barriers. The excellent chromosome morphology of these diploid species allows detailed cytological analysis of interspecific hybrids. Transgenic methodologies, well developed in the Solanaceae, allow the functional testing of candidate reproductive barrier genes as well as live imaging of pollen rejection events through the use of fluorescently tagged proteins. Proteomic and transcriptomics approaches are also providing new insights into the molecular nature of interspecific barriers. Recent progress toward understanding reproductive isolation mechanisms using these molecular and genetic tools is assessed in this review.