Utilizing behavioral experiments and chemical analyses, we examined whether pheromonally mediated sexual isolation exists between denning populations of red-sided garter snakes (Thamtnophis sirtalis parietalis) in Manitoba, Canada. Simultaneous choice tests conducted during the breeding season revealed that adult males from a hibernaculum in central Manitoba displayed a strong courtship preference for females from their own population over females from a hibernaculum in western Manitoba, whereas males from the western Manitoba hibernaculum showed no such preference. In addition. trailing experiments testing the response of males from the two hibernacula to familiar and unfamiliar female trails showed similar results, demonstrating that the observed male preference is mediated through chemical cues. Subsequent chemical analysis of the female sexual attractiveness pheromone. a homologous series of long-chain saturated and (omega-9 cis-unsaturated methyl ketones responsible for eliciting male courtship behavior and trailing behavior in garter snakes, showed significant variation in the composition of the pheromone between the two populations. Specifically, the two populations varied in the relative concentrations of individual unsaturated methyl ketones expressed by females. These results suggest that sexual isolation exists to a degree among denning populations of red-sided garter snakes due to variation in the expression of the female sexual attractiveness pheromone.