Viral, bacterial, parasitic and fungal pathogens pose a significant, current threat to global biodiversity. A virulent fungal pathogen (Pseudogymnoascus destructans; Pd) emerged in hibernating bats in eastern North America in 2006. In this paper, we seek to inform epidemiological models of the progression of Pd into populations of the little brown bat (Myotis lucifugus) in central Canada by characterizing the spatial genetic structure of the host ahead of the imminent arrival of Pd. We sampled 242 bats from eight hibernacula spanning 92,623 km2 and two ecozones. We genotyped all individuals at eight microsatellite loci and sequenced 300 bp of HVII in a subset (n = 72) to test the null hypothesis of contemporary panmixia. We found evidence of spatial genetic structure associated with ecozone boundaries, and a predominant north–west to south–east directionality of bat movements among hibernacula, which opposes the current approach of the pathogen. Our large study area (larger than the dispersal distance of individual bats) allowed us to detect the first evidence of contemporary population structure among hibernacula of M. lucifugus. Our results suggest that the potential spread of Pd into north-central Canada may be retarded by the opposing direction of gene flow of the host species, and our findings of directional gene flow can be used to inform management strategies for the spread of Pd into the area.