Detailed karyotype analysis including fluorescence in-situ hybridization with chromosome-specific and sub-regional probes revealed novel cytogenetic signatures for elucidating phylogenetic relationships in Vespertilionoidea. Two shared derived Robertsonian fusions characterize all members of this superfamily. Further, the majority of Molossidae species show karyotypes with 48 chromosomes, including six of seven species studied here, namely Ozimops petersi, Mormopterus jugularis, Tadarida teniotis, Mops mops, Molossus molossus and M. pretiosus. A shared derived feature of all molossid taxa studied is a variant homolog to Myotis myotis chromosome MMY22. Despite their overall similarity in karyotype composition with one large and three medium-sized bi-armed as well as 19 acrocentric to subtelocentric autosomal pairs, our detailed analysis revealed small differences which shed new light onto molossid phylogenetic relationships. Because of the retention of several ancestral chromosomal characters, O. petersi and M. jugularis represent early offshoots of the molossid tree. A unique derived paracentric inversion in the MMY6 homolog unites all other molossids studied so far. The next species to branch off is T. tadarida, followed by a group composed of M. mops and all studied New World taxa, united by a further derived inversion in the MMY5 homolog. In the light of our findings, we recommend to elevate the subspecies M. t. griseiventer to species rank, because the specimen from Venezuela with a 2n = 42 karyotype differed clearly from the nominate subspecies Molossops t. temminckii with 2n = 48. Further, karyotypes of Miniopterus and Natalus were studied. In the 2n = 46 chromosomal complement of the family Miniopteridae a large number of autapomorphic characters were found. Based on the observation of a common derived condition of the MMY22 homolog, we assume that long-winged bats (Miniopteridae) are closer related to vespertilionids than to molossids. Finally, karyotype comparison in Natalus revealed two autapomorphic characters in the 2n = 36 chromosomal complement.