Understanding social relationships and organization in colonial bat species can provide valuable insight into species ecology and potentially aid in conservation efforts of rare bat species. We applied social network analysis to describe social relationships and organization in 3 colonies of Rafinesque's big-eared bats (Corynorhinus rafinesquii) roosting in bottomland hardwood forests in Kentucky. We radiotracked 48 adult big-eared bats to 64 day-roosts over 549 bat-days during the summers of 2009–2011. We measured homophily, network centralization, density, transitivity, and core–periphery structure of networks of bats sharing common roosts, and we measured degree centrality of nodes (bats or roosts) within networks. Patterns of ties within each colony were homophilous by sex (E-I index = −0.87). Males were consistently the least central nodes in bat networks. Bat network centralization ranged from 1.2% to 40% among colonies, and roost network centralization ranged from 17% to 40%. The colony exhibiting the least centralized and most dense bat network also occupied habitat with low roost availability. This roost network was highly centralized, with bats frequently aggregating at a single roost. The colony with the most centralized and least dense bat network occupied habitat with a greater availability of roosts, resulting in diffuse networks of bats and roosts. Transitivity decreased after young became volant in the colony with highest roost availability. Our findings suggest that social structure in colonies of Rafinesque's big-eared bats is affected by the sex of individuals in colonies, reproductive season, and the preponderance of available day-roosting habitat.