Understanding factors that influence the spread of wildlife diseases can assist in designing effective surveillance programs and appropriate management strategies. Chronic wasting disease (CWD), a fatal prion disease of cervids, was detected in south-central Wisconsin in 2002 and over time has been identified increasingly farther west in the state leading to concerns about CWD spreading to Iowa. Our objective was to characterize genetic connectivity between white-tailed deer (Odocoileus virginianus) populations in eastern Iowa and western Wisconsin to assess the risk of CWD-infected deer dispersing to Iowa. We hypothesized that the Mississippi River, which separates the states, may restrict the movement of deer and thus disease. We genotyped hunter-harvested female deer collected from both states at 12 nuclear microsatellite loci (n = 249) and sequenced a portion of the mitochondrial DNA (mtDNA) control region (n = 173). Microsatellite data indicated there was low genetic differentiation (ΦPT = 0.005) between states and weak spatial genetic structure across the study area as a whole. Verifying expectations that dispersal in deer is male-biased, maternally inherited mtDNA data showed stronger spatial structuring across the study area and greater genetic differentiation between the states (ΦPT = 0.052) such that clustering analysis grouped the majority of deer from Iowa and Wisconsin into separate clusters. The low level of genetic differentiation between deer in northeast Iowa and southwest Wisconsin, primarily the result of dispersing males who have greater CWD prevalence than females, indicates that the Mississippi River is unlikely to prohibit the westward spread of CWD, and underscores the importance of continued CWD surveillance in Iowa. © 2011 The Wildlife Society.