Aegilops comosa Smith in Sibthorp et Smith, 1806 is diploid grass with MM genome constitution occurring mainly in Greece. Two morphologically distinct subspecies – Ae. c. comosa Chennaveeraiah, 1960 and Ae. c. heldreichii (Holzmann ex Boissier) Eig, 1929 are discriminated within Ae. comosa, however, genetic and karyotypic bases of their divergence are not fully understood. We used Fluorescence in situ hybridization (FISH) with repetitive DNA probes and electrophoretic analysis of gliadins to characterize the genome and karyotype of Ae. comosa to assess the level of their genetic diversity and uncover mechanisms leading to radiation of subspecies. We show that two subspecies differ in size and morphology of chromosomes 3M and 6M, which can be due to reciprocal translocation. Subspecies also differ in the amount and distribution of microsatellite and satellite DNA sequences, the number and position of minor NORs, especially on 3M and 6M, and gliadin spectra mainly in the a-zone. Frequent occurrence of hybrids can be caused by open pollination, which, along with genetic heterogeneity of accessions and, probably, the lack of geographic or genetic barrier between the subspecies, may contribute to extremely broad intraspecific variation of GAA_n and gliadin patterns in Ae. comosa, which are usually not observed in endemic plant species.