Most previous studies considering intraspecific differences in bat echolocation call frequency among geographic groups have related this to morphological features not directly involved in producing the characteristics of the emitted signal. We related a pattern of intraspecific call differences to the size of nasal chambers (estimated from radiographs of museum specimens), expecting chamber dimensions to be functionally coupled with the sound source. Such a relationship is potentially informative in the context of competing hypotheses that account for call frequency differences. Allopatry has been a precursor to differences in echolocation call frequency between isolated populations of the Australian endemic orange leaf-nosed bat (Rhinonicteris aurantia (Gray, 1845); Pilbara isolate: 120.99 ± 1.91 kHz; compared with Kimberley region: 114.65 ± 1.98 kHz and Northern Territory: 114.62 ± 2.10 kHz). Correlations with morphological features not directly involved in signal production or modification were either moderate (nose-leaf width) or absent (forearm length). Overall nasal volume was shown to be relatively smaller in the Pilbara population, which had higher average call frequency. This relationship was expected given the suggested function of nasal chambers in impedance matching. The finding is significant because nasal chamber size was the only character observed to vary in a species that was otherwise conserved morphologically, suggesting adaptation and not a simple scaling relationship with body size that might be more indicative of drift. We consider that the combination of patterns from echolocation call frequency and associated morphological features, as well as neutral DNA markers, provide adequate support for recognition of the separate populations of R. aurantia for conservation, according to more recent concepts that consider ecological as well as genetic characters when allocating groups to evolutionarily significant units.