A list of 139 specimens of bats belonging to 32 species of eight families originating from Zambia, housed in the collection of the National Museum, Prague, Czech Republic, is presented in a systematical review. The species lists are complemented by comments on distribution and morphometry data. The specimens represent 73 new records (species vs. locality) of bats from Zambia. The collection contains two species new for the Zambian fauna, Afropipistrellus grandidieri and Neoromicia somalica. Two species, Rhinolophus sakejiensis and Chaerephon bivittatus are documented for the second time from Zambia, the former bat for the first time after the species description at all. The record localities of Epomophorus labiatus, Rhinolophus mossambicus, and Neoromicia somalica shift margins of the whole known distribution ranges of these bats. In Epomophorus dobsonii, Nyctinomus aegyptiacus, Glauconycteris variegata, Pipistrellus rusticus, Scotophilus leucogaster, and S. viridis, the collection specimens represent new peripheral records making their distribution range margins more precise. Molecular genetic analysis revealed new distribution extent of particular mitochondrial lineages of the otherwise common species in Zambia, Hipposideros caffer, Nycteris thebaica, and Miniopterus natalensis s.str.
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