Abstract Recent studies suggest that roads can significantly impact bat populations. Though bats are one of the most threatened groups of European vertebrates, studies aiming to quantify bat mortality and determine the main factors driving it remain scarce. Between March 16 and October 31 of 2009, we surveyed road‐killed bats daily along a 51‐km‐long transect that incorporates different types of roads in southern Portugal. We found 154 road‐killed bats of 11 species. The two most common species in the study area, Pipistrellus kuhlii and P. pygmaeus , were also the most commonly identified road‐kill, representing 72 % of the total specimens collected. About two‐thirds of the total mortality occurred between mid July and late September, peaking in the second half of August. We also recorded casualties of threatened and rare species, including Miniopterus schreibersii , Rhinolophus ferrumequinum , R. hipposideros , Barbastella barbastellus , and Nyctalus leisleri . These species were found mostly in early autumn, corresponding to the mating and swarming periods. Landscape features were the most important variable subset for explaining bat casualties. Road stretches crossing or in the vicinity of high‐quality habitats for bats—including dense Mediterranean woodland (“montado”) areas, water courses with riparian gallery, and water reservoirs—yielded a significantly higher number of casualties. Additionally, more road‐killed bats were recorded on high‐traffic road stretches with viaducts, in areas of higher bat activity and near known roosts.