In the present study, we investigate the variation of European representatives of Alnus subg. Alnus, i.e., Alnus glutinosa and A. incana, in Europe and partly North Africa. Four distinct Alnus taxa were recognized using a multidisciplinary approach based on multivariate morphometrics, karyology, flow cytometry and nuclear microsatellite analyses. Besides the long-recognized diploid taxa (2n = 2x = 28) A. glutinosa and A. incana, we revealed the presence of morphologically, cytologically and genetically noticeably delimited tetraploid populations (2n = 4x = 56), which we consider new species. These form two geographically well-delimited clusters located in the Iberian Peninsula and the adjacent part of North Africa, designated here as Alnus lusi tanica, and in the Dinaric Alps extending south-easterly to Greece, denominated here as Alnus rohlenae. Detailed information concerning their diagnosis and geographical distribution is presented, and photographs of type specimens are provided.