Canada wild rye (CWR, Elymus canadensis L., 2n = 4x = 28) is a potential source of genes for disease resistance and environmental tolerance in barley (Hordeum vulgare L., 2n = 2x = 14). Tissue cultures were initiated from immature inflorescences of CWR × 'Betzes' barley hybrids to promote CWR introgression into barley through possible tissue culture induced chromosome breakage and exchange. Among the plants regenerated, some were missing one (2n = 20) or part of one (2n = 20 + telo) chromosome. The objective of this study was to identify the missing chromosome or chromosome arm in these regenerants through the analysis of molecular (RFLP) markers that previously had been mapped in barley. Forty-six hypoploid regenerants that traced to 30 separate explants obtained from 10 interspecific hybrid plants were evaluated. DNA was digested with the restriction enzyme HindIII, Southern blotted, and probed with 39 genomic and cDNA barley clones that identified sequences polymorphic between barley and CWR. Eight of these probes identified band loss patterns that separated the regenerants into two groups. One group, all with barley cytoplasm, were missing a CWR chromosome homoeologous to barley chromosome 3; a second group, all with CWR cytoplasm, were missing a CWR chromosome homoelogous to barley chromosome 7. These results indicated that chromosome elimination in culture was not random. The two cytoplasm groups were further differentiated by probes that identified band shifts. These band shifts were caused by differences in DNA methylation. Key words : Hordeum vulgare, aneuploidy, Elymus canadensis, tissue culture.