We investigated the extent of geographic variation in the echolocation calls of five species of microchiropteran Vespadelus in eastern Australia. Bat calls were recorded with Anabat II detectors and computers, and analysed using Analook software. A single call parameter, characteristic frequency (the frequency at the end of the flattest part of the call), was used to document changes in echolocation calls over geographic distance. For V. vulturnus and V. regulus, changes in call frequency were abrupt and sizeable (up to 14 kHz), with two or three frequency groups present within each species. In V. darlingtoni, the changes in frequency were gradual across their range, with an isolated island population resembling the closest region on the mainland. One species examined here, V. troughtoni, had calls that were consistent throughout its range. Calls of V. pumilus were also consistent across geographic regions except for at one site (Chichester State Forest). At this site calls occupied only the upper end of the species frequency range. Body size, measured as forearm length for each species, was significantly smaller at inland sites, but did not vary with latitude or consistently with intraspecific call variation. Broad patterns and possible causes of geographic variation in call frequency are discussed. We conclude that confident identifications of Vespadelus calls from the geographic regions outlined in this paper will only be made with reference calls collected from the relevant regions.