The blue mussels Mytilus edulis and Mytilus trossulus occur sympatrically and are able to hybridize in populations on the eastern coast of Newfoundland, Canada, presenting an opportunity to study their aggregational behavior. Aggregation behavior may therefore provide insight into post-settlement interactions and pre-zygotic reproductive isolation between the species. Three treatments were designed using M. edulis and M. trossulus to investigate their intraspecific and interspecific spatial distribution patterns. With Ripley’s K-function and Monte Carlo simulation analysis, we found that in the single-species treatment, M. edulis aggregated significantly but not M. trossulus. Based on results of two-way ANOVAs, both the number of aggregations and the moving distance were significantly affected by the treatments (single-species or mixed-species treatment) and times (24, 48, 72 and 96 h). In further pairwise comparisons using Tukey’s test, M. edulis aggregated differently with or without M. trossulus occupying the same tank, suggesting that the aggregational behavior of M. edulis could be driven by species-specific chemical cues. The result that M. edulis aggregates intraspecifically may increase the probability of intraspecific fertilization of the spawned gametes and thus function as a pre-zygotic reproductive isolation mechanism maintaining the blue mussel hybrid zone.