Comparatively little is known about hantavirus prevalence within rodent populations from the Midwestern US, where two species of native mice, the prairie deer mouse (Peromyscus maniculatus bairdii) and the white-footed mouse (Peromyscus leucopus noveboracensis), are dominant members of rodent communities. We sampled both species in central Indiana and tested individuals for presence of hantavirus antibodies to determine whether seroprevalence (percent of individuals with antibodies reactive to Sin Nombre virus antigen) differed between species, or among different habitat types within fragmented agro-ecosystems. Prevalence of hantavirus antibodies varied significantly between species, with seroprevalence in prairie deer mice (21.0%) being nearly four times higher than in white-footed mice (5.5%). Seroprevalence was almost eight times higher within the interior of row-crop fields (37.7%) occupied solely by prairie deer mouse populations, relative to field edges (5.2%) or adjacent forest habitat (6.1%). In the fragmented Midwestern agro-ecosystem of this study, prairie deer mice appear to be the dominant hantavirus reservoir, with particularly high seroprevalence in populations within the interior of row-crop fields.