Understanding population structure can lend insight into the spread of animal-borne disease, and the effects of anthropogenic land use on habitat. Raccoons are highly adaptive to human land development and can persist in a wide range of habitat types, making them ideal subjects for investigating the level of population structure in a highly fragmented area. A total of 323 raccoons were livetrapped from 7 locations encompassing 3 distinct habitat types (agriculture, urban forest preserves, and residential) across the Chicago metropolitan region (maximum distance between 2 sites was 128 km). Genetic analyses of 14 microsatellite loci indicate that although raccoon populations across the region share up to 50% of the allelic diversity, they segregated into at least 2 distinct subpopulations, dividing the Chicago metropolitan region into northern and southern groups with further structure occurring within these larger groups. Incorporating sample sites between the identified north–south groups may provide greater resolution as to where this split occurs. Although there is evidence of population structure between all sample sites, migrant analysis suggests there is enough gene flow to preserve genetic diversity throughout the population.