The large-footed myotis (Myotis macropus) is a specialist trawling bat with flexible roosting behaviour, being able to switch between caves, tree hollows and artificial roosts such as bridges, tunnels and culverts. However, little is known about how this species selects culvert roost sites in urban landscapes where hollows may be limited or absent. We surveyed 57 concrete culverts and found 21 M. macropus roosts comprising day and maternity roost sites; 305 bats were captured. Colony sizes averaged 20.6 ± 17.7 (range = 4–49) for maternity roosts and 2.0 ± 0.8 (range = 1–3) for day roosts. Roost culverts differed significantly from available culverts predominantly in terms of availability of microhabitat (lift holes and crevices). Roost culverts had lift holes that had greater cavity dimensions than available culverts and crevices were found only at roost culverts. Culverts containing microhabitat were a limited resource in this urban landscape and so increasing their availability may provide more urban roost sites for this specialist species.