Previous work has shown that chromosome pairing at metaphase I (MI) of wheat homologous chromosomes from different inbred lines (heterohomologous chromosomes) is reduced relative to that between homologous chromosomes within an inbred line (euhomologous chromosomes). In order to determine if a potential for this phenomenon exists in diploid species closely related to the wheat B genome, MI chromosome pairing was investigated between euhomologous and heterohomologous 6B(e) (=6S(e)) chromosomes, each from a different population of Aegilops longissima Schweinf. et Muschl. (2n = 2x = 14) substituted for chromosome 6B of Chinese Spring wheat (Triticum aestivum L., 2n = 6x = 42). Euhomologous and heterohomologous monotelodisomics, i.e., plants with one complete chromosome 6B(e) and a telosome of either 6B(e)p or 6B(e)q, were constructed in the isogenic background of Chinese Spring. Pairing at MI of the Ae. longissima chromosomes was reduced in heterohomologous monotelodisomics compared to that in the corresponding euhomologous monotelodisomics. The remaining 20 pairs of Chinese Spring chromosomes paired equally well in the euhomologous and heterohomologous monotelodisomics. Thus, the cause of the reduced pairing must reside specifically in the Ae. longissima heterohomologues. In the hybrids between the Ae. longissima lines that contributed the substituted chromosomes, pairing between the heterohomologous chromosomes was normal and did not differ from that of the euhomologous chromosomes. These data provide evidence that a potential for reduced pairing between the heterohomologues is present in the diploid species, but is expressed only in the polyploid wheat genetic background. The reduction in heterohomologous chromosome pairing was greater in the p arm than in the q arm, exactly as in chromosome 6B of wheat. It is concluded that the reduced pairing between Ae. longissima heterohomologues has little to do with constitutive heterochromatin. The value of chromosome pairing as an unequivocal means of determining the origin of genomes in polyploid plants is questioned.