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Southeastern myotis range in a broad band from Indiana and Illinois south along the Mississippi River and around the southeastern coastal plain to North Carolina. These bats are also reported in southeastern Virginia. Yet, despite this rather wide distribution, few maternity colonies have been discovered outside of Florida. This bat uses a variety of roosts in a variety of habitats including hollow trees in forested areas but also bridges, buildings, and culverts. Southeastern myotis are most commonly associated with caves, where they form large maternity colonies, sometimes containing thousands of individuals.
This bat appears to remain active for much of the year in the southern portions of its range, feeding on a large variety of aquatic insects. The extent to which this species relies upon forest resources is largely unknown, but recent radio-tracking studies have documented maternity colonies in areas of hardwood swamps, so perhaps their numbers are greater than once suspected in these areas. Some populations of southeastern myotis appear to be in steady decline and this bat is considered a Species of Concern by the U.S. Fish and Wildlife Service.