What is currently known about the ecology of North American hantaviruses has come largely from studies on Sin Nombre virus (SNV). We conducted a longitudinal study of Bayou virus (BAYV), the second-leading agent of hantavirus pulmonary syndrome in the United States. Antibodies to hantavirus were detected from Oryzomys palustris (most commonly infected species), Sigmodon hispidus, Peromyscus leucopus, Reithrodontomys fulvescens, and Baiomys taylori. However, only O. palustris had viral RNA in tissues and excreta, suggesting that antibodies detected in other species may have resulted from spill-over infection. Seroprevalence rates averaged around 16% for O. palustris and varied seasonally. The heaviest males exhibited the highest levels of seroprevalence. Seroprevalence was higher in coastal prairie (20.0%) than old-fields (10.5%) and was associated with host abundance. These patterns are similar to those of SNV and can be used in identification of potentially at-risk areas.