Little is known about geographic variation in the local composition of South American assemblages of mammals or the factors that may produce such variation. This is particularly unfortunate in Paraguay because it occurs at the interface of a number of phytogeographic regions (e.g., Cerrado, Chaco, Interior Atlantic Rainforest, and Pantanal) and is the point at which many temperate species reach their northern limits or at which tropical species reach their southern limits. Based on 2 years of intensive fieldwork, we documented the species and familial composition of bat assemblages at 25 sites throughout the country. We also estimated similarity among sites based on indices sensitive to presence or absence of species (Ochiai's index), to rank abundance of species (rank correlation index), or to relative abundance of species (Euclidean index). Geographic distance between sites accounted for little variation in composition based on presence–absence or rank abundances of species but accounted significantly for variation related to both identity and relative frequency of species. Distinct assemblages of species do not correspond to the 7 distinct biomes of the country; rather, strong differences exist between sites east (mesic) and west (xeric) of the Río Paraguay. For the most part, these differences are related to dominance by molossids (e.g., Eumops patagonicus, Molossops temminckii, and Molossus molossus) and vespertilionids (e.g., Lasiurus ega, Myotis albescens, and M. nigricans) in dry regions versus phyllostomids (e.g., Artibeus fimbriatus, A. lituratus, and Sturnira lilium) in mesic regions. As a consequence, classification of sites into 2 broad biogeographic regions based on bat familial composition generally is concordant with that based on plants.