Three species of long-eared bats (Plecotus auritus, P. macrobullaris and P. austriacus) are known to occur in sympatry in Western Europe. They share very similar morphological characters that complicate their reliable discrimination. As a consequence, many records from these areas are reported to the generic level only and the exact distribution of species is uncertain. Even cranial characters are ambiguous since discriminant values traditionally used to identify those species vary with geography. We present a novel use of molecular tools to identify reliably the three cryptic species of long-eared bats. The methods proposed here for time- and cost-effective molecular identification of Plecotus species are applied to variously degraded samples such as ancient museum specimens and guano samples collected under roosts. We used this molecular approach to identify over 810 samples and reassess the distribution range of the three species occurring in Switzerland. We further showed that some skull measurements used for species recognition overlap to a greater extent than anticipated and can be misleading for morphologically intermediate individuals.