Echolocation calls of four species of insectivorous bats of central Chile were recorded and characterized to determine vocal signatures that allow their identification in the field. Pulses of Tadarida brasiliensis were characterized by the highest duration and the lowest values for all frequencies, which do not overlap those of the remaining species. Tadarida emits narrowband, shallow frequency-modulated search calls. All three vespertilionid species studied (Histiotus montanus, Lasiurus varius and Myotis chiloensis) showed similar echolocation design to one another, consisting of a downward frequency modulation at the beginning of the signal followed by a narrowband quasi-constant frequency component; however, their calls differ by their spectral characteristics. Discriminant function analysis of six acoustic parameters (duration, initial frequency, slope frequency modulation, peak frequency, minimal and maximal frequencies) gave an overall classification of 87.4%, suggesting species could be correctly classified based on echolocation calls. Call duration and minimal frequency were the variables most important for species identification.