Patterns of intraspecific geographic variation of signaling systems provide insight into the microevolutionary processes driving phenotypic divergence. The acoustic calls of bats are sensitive to diverse evolutionary forces, but processes that shape call variation are largely unexplored. In China, Rhinolophus ferrumequinum displays a diverse call frequency and inhabits a heterogeneous landscape, presenting an excellent opportunity for this kind of research. We quantified geographic variation in resting frequency (RF) of echolocation calls, estimated genetic structure and phylogeny of R. ferrumequinum populations, and combined this with climatic factors to test three hypotheses to explain acoustic variation: genetic drift, cultural drift, and local adaptation. Our results demonstrated significant regional divergence in frequency and phylogeny among the bat populations in China's northeast (NE), central-east (CE) and southwest (SW) regions. The CE region had higher frequencies than the NE and SW regions. Drivers of RF divergence were estimated in the entire range and just the CE/NE region (since these two regions form a clade). In both cases, RF divergence was not correlated with mtDNA or nDNA genetic distance, but was significantly correlated with geographic distance and mean annual temperature, indicating cultural drift and ecological selection pressures are likely important in shaping RF divergence among different regions in China.