Islands are biodiversity hotspots, often containing numerous endemic species. This makes them also hotspots for conservation. Within the Mediterranean region, Sardinia is known for its comparatively high degree of endemism, including cryptic diversity. In this paper we aim to elucidate the variability of pipistrelles (Pipistrellus and Hypsugo) on Sardinia. More specifically, we ask which species occur on Sardinia and we describe the geographic affiliations of these evolutionary lineages. We sequenced ca. 560 bp of the 16S rRNA gene from 36 pipistrelle specimens representing 17 localities from all major parts of Sardinia. For comparison we added samples from the entire Mediterranean region as well as sequences stored at GenBank. We constructed Bayesian phylogenetic trees and minimum spanning networks to identify which species occur on Sardinia and to infer their genetic affiliation to lineages occurring throughout the Mediterranean and the Canary Islands. We identified five pipistrelle lineages on Sardinia: Pipistrellus pipistrellus (haplolineage II), P. pygmaeus, P. kuhlii/desertii, Hypsugo savii s.str. and H. cf. darwinii. Colonization of Sardinia occurred at different times from different geographical sources, namely Europe and Africa. Some lineages may have invaded Sardinia recently. The Sardinian H. cf. darwinii may be endemic to the island. Our results highlight the importance of Sardinia as a major Mediterranean hotspot for bat biodiversity. The island harbours a pipistrelle diversity that is higher than that on any other Mediterranean island. Lying geographically at the interface between Europe and Africa, Sardinia combines elements from both continents.