ABSTRACT. Aegilops biuncialis can be hybridized with wheat (Triticum spp) and has been used for wheat breeding and genetic studies. The , with a karyotype formula of 2n = 4x = 28 = 14m + 10sm + 4st. Fluorescence in situ hybridization (FISH) and C-banding approaches were used to analyze the A. biuncialis accession chromosomes at the mitotic stage. Based on the C-banding and FISH patterns, all U b and M b chromosomes could be discriminated simultaneously; the three A. biuncialis accessions exhibited similar patterns, suggesting a common origin. The U b genome from A. biuncialis resembled the U genome in the diploid species A. umbellulata, and it may be related to the tetraploid species containing the U genome. The M b genome had some differences compared to the M genome in the diploid species A. comosa, and it may be related to the tetraploid species possessing the M genome. A generalized ideogram was proposed for the A. biuncialis genome, which could be useful for standardized and accurate identification of the A. biuncialis karyotype and chromosomes.