The origin and meaning of echolocation call frequency variation within rhinolophid bats is not well understood despite an increasing number of allopatric and sympatric examples being documented. A bimodal distribution of mean regional call frequency within the Okinawa-jima Island population of Rhinolophus cornutus pumilus (Rhinolophidae) provided a unique opportunity to investigate geographic call frequency variation early in its development. Individual resting echolocation frequencies, partial mitochondrial DNA D-loop sequences and genotypes from six microsatellite loci were obtained from 288 individuals in 11 colonies across the entire length of the island, and nearby Kume-jima Island. Acoustic differences (5–8 kHz) observed between the north and south regions have been maintained despite evidence of sufficient nuclear gene flow across the middle of the island. Significant subdivision of maternally inherited D-loop haplotypes suggested a limitation of movement of females between regions, but not within the regions, and was evidence of female philopatry. These results support a ‘maternal transmission’ hypothesis whereby the difference in the constant frequency (CF) component between the regions is maintained by mother–offspring transmission of CF, the restricted dispersal of females between regions and small effective population size. We suggest that the mean 5–8 kHz call frequency difference between the regions might develop through random cultural drift.