Allium subgenus Melanocrommyum is a species-rich group of perennial onions with uniform karyology, ecology, and breeding system. Thus, the cytological, ecological, and physiological factors often correlated with genome size should have negligible effect on genome size variation in Melanocrommyum. We measured DNA content in subgenus Melanocrommyum using flow cytometry based on propidium iodide staining and analyzed the evolution of genome size in a phylogenetic context. The observed 2C genome size variation in 160 accessions of 70 species of the subgenus was high, varying from 26.26–78.73 pg. The significant phylogenetic signal in genome size data suggests that distribution of genome size is in accordance with phylogenetic clades identified by the analysis of nuclear ITS sequences. Estimation of ancestral genome sizes using generalized least squares revealed lineages with increasing as well as decreasing DNA content. We found within-species genome size variation to be mostly below 2.5%. In species where intraspecific genome size differences were in a range of 6–9%, we suggest the existence of cryptic species, as previously inferred by molecular markers. Thus, genome size variation reflects incipient speciation or diversification in Allium subgenus Melanocrommyum. About two-fold differences in DNA content in several Melanocrommyum species indicate the occurrence of diploid and tetraploid cytotypes in these taxa, which for some species has been confirmed by chromosome counts.