The two widespread sibling species of pipistrelle bats, the soprano pipistrelle (Pipistrellus pygmaeus), and the common pipistrelle (Pipistrellus pipistrellus), that emit at distinct call frequencies (maximum energy at around 55 and 45 kHz respectively), have been found in sympatry over much of Europe, but little is known about their relative abundance and possible habitat use. In this study, we provide the first record of P. pygmaeus in southern Switzerland, confirmed by echolocation analysis and mitochondrial DNA sequencing. During 70 catching sessions in the southern region of the Swiss Alps, echolocating bat calls were recorded to define the distribution/presence of the two species. Habitat characteristics of capture sites suggested that the sibling species have a high degree of niche overlap (0.88). Nevertheless, P. pygmaeus seemed to select agricultural areas and riversides more frequently than P. pipistrellus, which is found more often in urban areas. Divergence in the cytochrome b gene of mitochondrial DNA of 37 bats confirmed the echolocation data for 31 of 33 P. pipistrellus and for two of four P. pygmaeus, while four animals, two of each species, had been mis-classified based on echolocation only. A canonical discriminant function using morphological data was not reliable to distinguish the sibling species.