Specialist species face higher extinction risks as a result of smaller, isolated populations with reduced gene flow. The large-footed myotis (Myotis macropus) is the only microbat in Australia specialised for foraging directly over water surfaces. Such highly specialised feeding ecology restricts the distribution of M. macropus to coastal regions and inland waterways. Using five novel and two existing nuclear microsatellite markers, we investigated genetic diversity within and among five M. macropus populations in Victoria. Significant genetic differentiation was detected between all populations. FST values between populations ranged from 0.02 to 0.24. We suggest that the movement of M. macropus throughout the landscape is constrained by the availability of permanent waterways and associated riparian habitats. These findings represent important considerations for the conservation of this specialist species and the management of riparian vegetation, particularly on private land.