On the North American Great Plains, several snake species reach their northern range limit where they rely on sparsely distributed hibernacula located in major river valleys. Independent colonization histories for the river valleys and barriers to gene flow caused by the lack of suitable habitat between them may have produced genetically differentiated snake populations. To test this hypothesis, we used 10 microsatellite loci to examine the population structure of two species of conservation concern in Canada: the eastern yellow-bellied racer (Coluber constrictor flaviventris) and bullsnake (Pituophis catenifer sayi) in 3 major river valleys in southern Saskatchewan. Fixation indices (FST) showed that populations in river valleys were significantly differentiated for both species (racers, FST = 0.096, P = 0.001; bullsnakes FST = 0.045-0.157, P = 0.001). Bayesian assignment (STRUCTURE) and ordination (DAPC) strongly supported genetically differentiated groups in the geographically distinct river valleys. Finer-scale subdivision of populations within river valleys was not apparent based on our data, but is a topic that should be investigated further. Our findings highlight the importance of major river valleys for snakes at the northern extent of their ranges, and raise the possibility that populations in each river valley may warrant separate management strategies.