Methodological improvements now allow routine analyses of highly degraded DNA samples as found in museum specimens. Using these methods could be useful in studying such groups as rodents of the genus Gerbillus for which i) the taxonomy is still highly debated, ii) collection of fresh specimens may prove difficult. Here we address precise taxonomic questions using a small portion of the cytochrome b gene obtained from 45 dry skin/skull museum samples (from 1913 to 1974) originating from two African and three Asian countries. The specimens were labelled G. gerbillus, G. andersoni, G. nanus, G. amoenus, G. perpallidus and G. pyramidum, and molecular results mostly confirmed these assignations. The close relationship between G. nanus (Asian origin) and G. amoenus (African origin) confirmed that they represent vicariant sibling species which differentiated in allopatry on either side of the Red Sea. In the closely related G. perpallidus and G. pyramidum, specimens considered as belonging to one G. pyramidum subspecies (G. p. floweri) appeared closer to G. perpallidus suggesting that they (G. p. floweri and G. perpallidus) may represent a unique species, distributed on both sides of the Nile River, for which the correct name should be G. floweri. Furthermore, the three other G. pyramidum subspecies grouped together with no apparent genetic structure suggesting that they may not yet represent genetically differentiated lineages. This study confirms the importance of using these methods on museum samples, which can open new perspectives in this particular group as well as in other groups of interest.