Baylisascaris procyonis, the raccoon roundworm, is increasingly being recognized for its zoonotic and public health importance. Fine-scale analyses of the population genetics of this species have been hampered due to a lack of appropriate genetic markers. To this end, we developed 8 novel polymorphic microsatellites for B. procyonis and used these markers to elucidate microgeographic structuring of this parasite in a 500-km2 study area in western Michigan. Our analyses revealed significant levels of genetic differentiation amongst the 74 worms collected from 10 different raccoons. Critically, Bayesian clustering indicated 2 genetically distinct groups, one on either side of the Grand River which bisects our study area. Estimates of FST, and results from AMOVA and isolation by distance, further corroborated a scenario whereby the river is acting as a barrier to gene flow, a rather unexpected finding given the high vagility of raccoons and microgeographic scale of the analysis. It is possible that the Grand River is a major dispersal barrier for B. procyonis because raccoons are most likely to disperse across the river when it is frozen, and worm burden in raccoons approaches zero during the winter.