Raccoons (Procyon lotor) and Virginia opossums (Didelphis virginiana) are highly abundant, synanthropic mesocarnivores that can transmit epizootic diseases of critical importance to human and livestock health such as rabies and tuberculosis. Individuals of both species can exhibit large dispersal distances and our understanding of population connectivity for both of these species is limited. Data on population connectivity and potential for disease transmission are needed for each of these species for the development of data-informed management, control, and vaccination programs. Genetic connectivity among populations can be estimated by evaluation of metrics such as of isolation by distance (IBD) signatures and population structure. Such metrics are influenced not only by geographic distance and barriers to dispersal but also by the life-history characteristics of the species in question. We investigated the scale at which these species exhibited evidence of connectivity via gene flow, using samples collected from 9 sites spanning 254 km across north-central, west-central, south-central, and southern Indiana, USA. We evaluated genetic connectivity and signatures of IBD using Bayesian clustering analyses and matrix correlation methods. In the Bayesian model, raccoons were assigned to 3 genetic clusters, whereas Virginia opossums exhibited apparent panmixia. Spatial autocorrelation results indicated a significant positive correlation between genetic and geographic distance for raccoons at distances up to 25 km and for Virginia opossums up to 4 km. Interspecific differences in reproductive biology, social behavior, and dispersal are likely the causes for differences between these 2 species in the spatial partitioning of populations across broad ranges. In addition, geographic features and past glaciation events may be affecting the genetic characteristics of present-day raccoon populations. Improved information regarding interpopulation distance for these 2 synanthropic species may contribute to more effective management and disease-control programs. © 2014 The Wildlife Society.