Aegilops biuncialis can be hybridized with wheat (Triticum spp) and has been used for wheat breeding and genetic studies. The A. biuncialis karyotype (U(b) U(b) M(b) M(b)) was investigated based on three A. biuncialis accessions grown in China. Two pairs of SAT chromosomes were identified as 1U(b) and 5U(b), 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.