A revision of the genus Gymnospermium (Berberidaceae) in the Balkan Peninsula is carried out. Three species are recognised. Gymnospermium maloi is described as a new species from Mt. Picari in Gjirokastra district, southern Albania. It is compared with the closely related G. scipetarum which has a different habitat and distribution in central Albania and southern Montenegro. The chromosome number and karyotype features of G. maloi are provided for the first time. The chromosome formula of 2n = 2x = 14 (1 metacentric, 1 meta-submetacentric and 5 submetacentric chromosome pairs) is unusual as 2n = 16 has been reported for other members of the genus. The nuclear DNA content (2C-value) of all three species was determined. The genome size of G. maloi is 29.44 (± 0.47) pg, for G. scipetarum (chromosome number still unknown) 29.55 (± 1.35) pg, and for G. peloponnesiacum (2n = 2x = 16) 31.93 (± 2.38) pg. These values are the first genome size measurements for the genus. All three species are mapped and fully illustrated. A key to the European species is also presented.