Using isozyme analysis, we studied the genetic diversity among 316 Bromus hordeaceus (Poaceae) accessions of different geographic origin and belonging to five subspecies. Eight putative isozyme loci revealed remarkable allozyme variation. The isozyme variation occurred as different fixed homozygous and heterozygous phenotypes without segregation among the seed progeny. We detected no subspecies-specific allozymes. Two isozymes tend to differentiate among subspecies, indicating that subspecies of B. hordeaceus are still undergoing contemporary process of divergence. The differentiation into 65 multilocus isozyme lineages (MLILs) was not related to the geographic origin of B. hordeaceus accessions. Bromus hordeaceus did not lose genetic variation towards its range margins: we found 7.7% out of total MLILs in the northernmost part of a core distribution range and 7.7%–40% in the southern part. We recorded silencing of one homeoallelic isozyme gene in the allotetraploid genome of B. hordeaceus for accessions of different geographic origins.