Abstract The mechanisms used to facilitate mate location among insects—such as pheromones—can inhibit interspecific attraction and confer reproductive isolation. However, pheromone components seem conserved within the genus Monochamus (Coleoptera: Cerambycidae) with the compound 2-(undecyloxy)ethanol (referred to as monochamol) being identified as the sex-aggregation pheromone or putative pheromone attractant for at least 15 species in this genus. This pheromone parsimony suggests the existence of additional isolating mechanisms. Here we describe the results of laboratory and field assays of additional mechanisms, including laboratory volatile collections and field trapping experiments that assessed the potential presence of additional pheromone components, diel rhythms of pheromone production, and phenological/diel flight activity in the reproductive isolation of sympatric populations of three species of Monochamus in the Great Lakes Forest Region of Ontario, Canada. Chemical analyses of volatile extractions indicate that monochamol is produced by male Monochamus maculosus and Monochamus scutellatus, but no qualitative differences were observed in male extracts of these two species suggesting that there are no additional pheromone components that confer specificity. No quantitative differences were found in the production of monochamol by male M. scutellatus during the photophase and scotophase suggesting there is no diel rhythm in pheromone production. Our results indicate that M. scutellatus flies earlier in the season and day than M. maculosus and Monochamus notatus, which could partially contribute to their reproductive isolation. Overall, no obvious differences in pheromone composition were observed but minor differences in flight times were observed, suggesting other isolating mechanisms may exist.