Despite only emerging in the past decade, white-nose syndrome has become among the most devastating wildlife diseases known. The pathogenic fungus Pseudogymnoascus destructans infects hibernating bats and typically leads to high rates of mortality at hibernacula during winter in North America. We developed a set of genetic markers to better differentiate P. destructans isolates. We designed and successfully characterized these 23 microsatellite markers of P. destructans for use in disease ecology and epidemiology research. We validated these loci with DNA extracted from a collection of P. destructans isolates from the US and Canada, as well as from Europe (the likely introduction source based on currently available data). Genetic diversity calculated for each locus and for the multilocus panel as a whole indicates sufficient allelic diversity to differentiate among and between samples from both Europe and North America. Indices of genetic diversity indicate a loss of allelic diversity that is consistent with the recent introduction and rapid spread of an emerging pathogen.