Echolocation calls geographical differences and their causes are fundamental researches and critical areas of animal ecology. It has important scientific significance in exploration speciation, biodiversity protection, and animal survival mechanism. In this study, we investigated the differences of echolocation calls structure among geographical populations of least horseshoe bat (Rhinolophus pusillus) from a relative large scale, including nine geographic populations: Hainan (Haikou), Hebei (Handan), Shandong (Mengyin), Jiangsu (Yixing), Guangxi (Guilin), Guangdong (Shaoguan, Shenzhen, Zhuhai) and Macau by t test and One-way ANOVA. Furthermore, we would explore the factors that cause the sound differences among different geographical populations by Pearson correlation. The results showed that females were slightly larger than males (FA, females: 38.12±0.26 mm, males: 36.79±0.32 mm, t=-2.75, P<0.01), and the dominant frequency of females (109.90 ± 0.31 kHz) was higher than that of males (male: 108.47 ± 0.15 kHz) (t = -4.33, P <0.01) (Table 1). The echolocation calls among different geographical populations shown a certain degree variations in pulse duration, pulse interval, frequency, and bandwidth. Forearm length and body mass of females were both negatively correlated with their dominant frequency (FA: r=-0.281, P=0.032; Mass: r=-0.371, P=0.004) (Fig 2 and Fig 3), and the rainfall and the dominant frequency were positively correlated (r=0.853, P=0.007) (Fig 4). But the echolocation calls of males have no correlations with forearm length, body mass, and rainfall. In additionally, the echolocation parameters of both males and females have no correlations with geographical distance, temperature, and humidity. These results showed that the echolocation calls varied among different geographic populations of horseshoe bat, which might be mainly effected by local habitats, such as rainfall. This variation exhibits adaptive evolution when the animals live in different habitats.