One of the North American bat species most impacted by white-nose syndrome (WNS) is the northern long-eared bat Myotis septentrionalis, which as a result has been listed under the Endangered Species Act. WNS was first detected in Wisconsin in 2014. Unfortunately, little is known regarding the ecology of M. septentrionalis in this state pre-WNS to guide management supporting post-WNS recovery efforts. The objectives of our research were to (1) assess characteristics of trees that are associated with roost tree selection and (2) investigate how characteristics of maternity colony networks compare to colonies in the eastern USA. We mist-netted at 3 sites in Wisconsin in 2015 and 2016, and affixed radio transmitters to 39 female M. septentrionalis. We tracked bats to 53 confirmed day roosts. We found that roost trees were larger, more decayed, and more likely to be in dominant canopy closure areas than random trees. Oaks Quercus spp. were used most frequently and in proportion to their availability in the landscape at 2 field sites, whereas invasive black locust Robinia pseudoacacia was used more than expected based on availability at another site. Overall, minimum convex polygon sizes for maternity roosts were variable (5.2 to 8.9 ha) but similar to values reported for other regions. However, network centrality was low, indicating equitable use of day roosts and more frequent roost switching compared to other regions. Our findings provide information that increasing availability of potential day roosts in the landscape during the reproductive period may improve recruitment, which may in turn mitigate some of the detrimental population effects from WNS.