The identification of two cryptic bat species of the genus Pipistrellus using a non-destructive and quick method of multiplex PCR and agarose gel electrophoresis is described. Two primer combinations were able to produce species-specific bands that identified reliably individuals that were previously identified by mtDNA sequencing. Robustness of the method was subsequently successfully tested on 16 randomly selected free-living animals from central Europe (tissue samples obtained from a 3 mm punch of wing-membrane) identified to species on the basis of echolocation calls. Nine out of 15 museum specimens and 100% of fresh faecal samples from seven individuals were also successfully identified by this method. The described method thus provides a good way to routinely distinguish two Pipistrellus species by using non-destructive sampling of living individuals or droppings, and will be used in field studies of their ecology.