Geographical isolation may lead to the populations having different ecological characteristics. Geographic isolation have been known to cause echolocation call differences in bats. In order to look at geographic differences of echolocation calls of Great Horseshoe Bats (Rhinolophus ferrumequinum), populations of inland and Jeju island habitats were examined in Korea. The study areas were some abandoned mines in the inland and caves of Jeju island, which were known as rest sites of Great Horseshoe Bats during the active season. Recording was done in two ways: Hand-held, Free-flying. Recording pulse was analyzed into five parameters: Maximum Frequency (FMAX), Minimum Frequency (FMIN), Peak Frequency (PF), Duration (D), Inter pulse Interval (IPI). Interestingly, The present study shows that the difference in echolocation between the inland and island populations. The PF of bats inhabited inland was 69 kHz. but, the PF of bats inhabited island (Jeju) was 71 kHz. There was a difference between regions. Discriminant analyses also showed clear difference between the inland and the island populations. Especially, PF of Korea population is lower than that of Europe (82 kHz) and higher than that of Japan (65 kHz).