Until recently, eastern woodrats (Neotoma floridana, Ord) at Pine Hills, Union County, were believed to be the only extant population in Illinois. During 1994, additional populations in adjacent Jackson County were discovered at Fountain Bluff, Horseshoe Bluff, and Little Grand Canyon. Genetic variation among Illinois woodrat populations was estimated based on microsatellite DNA at 6 loci. The four populations were separated by 2–14 km, yet all exhibited significant genetic differentiation. Fountain Bluff was the most geographically isolated population and had the lowest genetic heterozygosity. A sample of individuals from a more contiguous population in Missouri exhibited the highest heterozygosity. Populations were monitored by mark-recapture live- trapping for 39 months at Pine Hills and 19 months at Fountain Bluff. Although the Fountain Bluff population was smaller than the Pine Hills population, seasonal demographics and reproductive output were similar. Natural dispersal from all 4 populations was limited by fragmented ahabitat, natural barriers, and anthropogenic barriers. Reintroductions of woodrats may be necessary for the species to repopulate formerly occupied sites throughout southern Illinois.