Assessing the impact of forest management on bat communities requires a reliable method for measuring patterns of habitat use by individual species. A measure of activity can be obtained by monitoring echolocation calls, but identification of species is not always straightforward. We assess the feasibility of using analysis of time-expanded echolocation calls to identify free-flying bats in the Tomakomai Experimental Forest of Hokkaido University, Hokkaido, northern Japan. Echolocation calls of eight bat species were recorded in one or more of three conditions: from hand-released individuals, from bats flying in a confined space and from bats emerging from their roost. Sonograms of 171 calls from 8 bat species were analyzed. These calls could be categorized into 3 types according to their structure: FM/CF/FM type (Rhinolophus ferrumequinum), FM types (Murina leucogaster, Murina ussuriensis, Myotis macrodactylus and Myotis ikonnikovi) and FM/QCF types (Eptesicus nilssonii, Vespertilio superans and Nyctalus aviator). Sonograms of the calls of R. ferrumequinum could easily be distinguished from those of all other species by eye. For the remaining calls, seven parameters (measures of frequency, duration and inter-call interval) were examined using discriminant function analysis, and 92% of calls were correctly classified to species. For each species, at least 80% of calls were correctly classified. We conclude that analysis of echolocation calls is a viable method for distinguishing between species of bats in the Tomakomai Experimental Forest, and that this approach could be applied to examine species differences in patterns of habitat-use within the forest.