Intraspecific variability in echolocation calls could be an important factor hampering the accurate acoustic identification of species in the field. We studied variations in the echolocation behavior of Lasiurus varius in relation to habitat structure. Echolocation calls of L. varius reflected the degree of clutter present in its foraging areas. Bats foraging in an uncluttered habitat emitted longer and lower-bandwidth calls, with the lowest frequency values, whereas bats foraging in a cluttered habitat broadcast shorter and broadband signals, showing an increase in frequency content of their calls. Discriminant function analysis gave an overall classification of 76% of the calls emitted in the different flight situations. Our results highlight the need for independent recordings at each study area or habitat type to circumvent potential echolocation call variations, particularly in fragmented landscapes.