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.