Based on the co-linearity in the Triticeae, comparative RFLP analysis of 2M chromosome of Ae. comosa Sibth et Sm. was performed with 2MS and 2M additional lines of Triticum aestivum L. cv. Chinese Spring. Among the wheat RFLP probes conserved in the short arms of wheat chromosome 2, those above psr912 were located on the long arms of 2M in Aegilops comosa. The rest probes on the short arm and all the probe sequences on the long arm of group 2 chromosome in wheat were conserved on the equivalent chromosomal position in Aegilops comosa. So, it is apparent that some chromosomal segment from the short arm had been transferred to long arm while reconstructing 2M chromosome relative to wheat chromosomes. The break-point was located between psr912 and psr131 of the short arm. This rearrangement of chromosome 2M might be a molecular evidence of the M genome speciation from an ancestral type.