Hesperis tosyaensis A.Duran, a new species from northern Anatolia, is described and illustrated. It grows under open Quercus forest. The new species is distinct from the closely allied H. ozcelikii A.Duran by the following features: stem erect, 45-90 cm tall, hispid, hairs 2-2.5 mm, and fruit glabrous. Genetic differentiation between closely related Hesperis L. taxa and Matthiola W.T.Aiton was examined by molecular analyses of the ISSR. The chromosome number for Hesperis tosyaensis is 2n = 14. Information is also presented on its ecology and conservation status. In addition, Hesperis aspera E.Fourn. is treated as a synonym of Hesperis bicuspidata (Willd.) Poir.