Landscape features influence wildlife movements across spatial scales and have the potential to influence the spread of disease. Chronic wasting disease (CWD) is a fatal prion disease affecting members of the family Cervidae, particularly white-tailed deer (Odocoileus virginianus), and the first positive CWD case in a wild deer in Ohio, USA, was recorded in 2020. Landscape genetics approaches are increasingly used to better understand potential pathways for CWD spread in white-tailed deer, but little is known about genetic structure of white-tailed deer in Ohio. The objectives of our study were to evaluate spatial genetic structure in white-tailed deer across Ohio and compare the support for isolation by distance (IBD) and isolation by landscape resistance (IBR) models in explaining this structure. We collected genetic data from 619 individual deer from 24 counties across Ohio during 2007–2009. We used microsatellite genotypes from 619 individuals genotyped at 11 loci and haplotypes from a 547-base pair fragment of the mitochondrial DNA control region. We used spatial and non-spatial genetic clustering tests to evaluate genetic structure in both types of genetic data and empirically optimized landscape resistance surfaces to compare IBD and IBR using microsatellite data. Non-spatial genetic clustering tests failed to detect spatial genetic structure, whereas spatial genetic clustering tests indicated subtle spatial genetic structure. The IBD model consistently outperformed IBR models that included land cover, traffic volume, and streams. Our results indicated widespread genetic connectivity of white-tailed deer across Ohio and negligible effects of landscape features. These patterns likely reflect some combination of minimal resistive effects of landscape features on white-tail deer movement in Ohio and the effects of regional recolonization or translocation. We encourage continued CWD surveillance in Ohio, particularly in the proximity of confirmed cases.