Identifying natural barriers to movements of hosts associated with infectious diseases is essential for developing effective control strategies. Raccoon rabies variant (RRV) is a zoonosis of concern for humans because its main vector, the raccoon (Procyon lotor), is found near residential areas. In Québec, Canada, all cases of RRV found in raccoons since 2006 were detected on the eastern side of the Richelieu River, suggesting that this river acts as a barrier to gene flow and thus, the potential for RRV to spread. The objectives of this study were to characterize the genetic structure of raccoon populations and assess the effect of the Richelieu River on the population structure in southern Québec, Canada. We also evaluated whether RRV spread potential differed between sex and at a larger spatial scale. Our analyses revealed a weak signal of genetic differentiation among individuals located on each side of the Richelieu River. At a larger spatial scale, genetic structuring was weak. Our results suggest that rivers might not always efficiently restrain raccoon movements and spread of RRV. We suggest that the difference in genetic structure found between sexes can be partly explained by male movements during the breeding season in winter, when ice bridges allow passage over most rivers in Québec.