Hantaviruses may cause serious disease when transmitted to humans by their rodent hosts. Since their emergence in the Americas in 1993, there have been extensive efforts to understand the role of environmental factors on the presence of these viruses in their host rodent populations. HPS outbreaks have been linked to precipitation, but climatic factors alone have not been sufficient to predict the spatial-temporal dynamics of the environment-reservoir-virus system. Using a series of mark-recapture sampling sites located at the Mbaracayú Biosphere Reserve, an Atlantic Forest site in eastern Paraguay, we investigated the hypothesis that microhabitat might also influence the prevalence of Jaborá hantavirus within populations of its reservoir species, Akodon montensis. Seven trapping sessions were conducted during 2005–2006 at four sites chosen to capture variable microhabitat conditions within the study site. Analysis of microhabitat preferences showed that A. montensis preferred areas with little forest overstory and denser vegetation cover on and near the ground. Moreover, there was a significant difference in the microhabitat occupied by antibody-positive vs antibody-negative rodents, indicating that microhabitats with greater overstory cover may promote transmission and maintenance of hantavirus in A. montensis.