The variability of the external characters of four sibling Plecotus species in Croatia was analysed. For the recently discovered species P. macrobullaris and P. kolombatovici we used specimens identified by mitochondrial DNA sequences as key specimens. Living individuals of P. kolombatovici can be distinguished from P. macrobullaris and P. auritus by means of shorter thumb and hind foot, more clearly than distinguishing P. austriacus from P. auritus. Using the triangular pad on the lower lip it is easy to distinguish P. macrobullaris from all other species in the field. Sympatric distribution is confirmed for P. macrobullaris and P. auritus as well as for P. auritus and P. austriacus. In Istria, P. kolombatovici was found only at one site syntopic with P. austriacus and at another site inhabiting the same church attic with P. macrobullaris.