The recording and analysis of echolocation calls are fundamental methods used to study bat distribution, ecology, and behavior. However, the goal of identifying bats in flight from their echolocation calls is not always possible. Unlike bird songs, bat calls show large variation that often makes identification challenging. The problem has not been fully overcome by modern digital-based hardware and software for bat call recording and analysis. Besides providing fundamental insights into bat physiology, ecology, and behavior, a better understanding of call variation is therefore crucial to best recognize limits and perspectives of call classification. We provide a comprehensive overview of sources of interspecific and intraspecific echolocation call variations, illustrating its adaptive significance and highlighting gaps in knowledge. We remark that further research is needed to better comprehend call variation and control for it more effectively in sound analysis. Despite the state-of-art technology in this field, combining acoustic surveys with capture and roost search, as well as limiting identification to species with distinctive calls, still represent the safest way of conducting bat surveys.