The geographical variation in bat echolocation calls is common. However, it is not clear whether juveniles of bats are able to discriminate the echolocation differences between populations before the juveniles′ diffusion. In this study, we used two-choice playback experiments to detect the discrimination ability and behavioural response of juveniles (one-month old) to geographical variation in echolocation calls of the Greater Horseshoe Bats (Rhinolophus ferrumequinum). We used four response parameters to measure the behaviour of juveniles towards to either local or foreign echolocation calls: number of flights, time spent in flight, number of landings, and time spent in detection. The result showed that there was a significant difference in number of flights, time spent in flight and time spent in detection by Mann-Whitney U test (P < 0.05), but no any significant difference in number of landings (Fig. 2) was detected. Overall, the juveniles flew more often and spent more time in flying to detect the calls made by local population than those made by the foreign populations (Fig. 2, Table 1). We performed a principal component analysis for the four response parameters of juveniles and found out that there were statistically significant differences in the PC1 scores between playback treatments (P < 0.01) for the result of Mann-Whitney U tests. The juveniles responded more strongly to local than to foreign echolocation calls (PC1: t10 = 5.25, P < 0.001; PC2: t10 = 2.34, P < 0.05) by the paired samples Student′s t tests. This study showed that the juveniles of the Greater Horseshoe Bats was capable to discriminate the acoustic difference between their own population and other population.