Sexual isolation, the reduced ability of organisms of different species to successfully mate, is one of the reproductive barriers that prevent gene flow between different taxa. Various species-specific signals during courtship are attributed to the sexual isolation between them. Drosophila pseudoobscura has been widely used to study the behavioral and genetic mechanisms underlying selection for sexual isolation, as a model system for speciation. D. pseudoobscura and its sibling species, D. persmilis, live together in many locations but are reproductively isolated from one another. North American geographic strains of D. pseudoobscura from the American West mate at random. Several decades ago, D. pseudoobscura was collected in the vicinity of Bogota, Colombia, and later named the subspecies D. pseudoobscura bogotana. Nearly 5,000 matings were observed in this study. We analyzed mating behavior and cuticular hydrocarbon profiles as well as courtship within and between North American and Bogota strains of D. pseudoobscura. Here we report for the first time that Bogota strains of D. pseudoobscura do show statistically significant sexual isolation from North American strains. In addition, there are quantitative variations in cuticular hydrocarbons as well as in courtship behavior between Bogota and North American strains, and females of both North American and Bogota strains show strong preference for North American strain males having high mating propensities, suggesting that the Bogota strains are at an early stage that could lead to a separate species.