Dasypyrum villosum (L.) Candargy (2n=14, V genome) is a wild, allogamous, diploid grass species that is a potential genetic resource for wheat improvement. The diversity of high-molecular-weight (HMW) glutenin subunits of the seed storage proteins of this species was examined in populations sampled in their natural habitats in Italy and Yugoslavia where the species is widely distributed. The results of selfed progeny tests confirmed that the allelic variation of HMW-glutenin subunits in D. villosum is controlled by a single locus (Glu-V1). Fourteen alleles at Glu-V1 were found among 982 individuals representing 12 populations from Italy and two from Yugoslavia, with a mean of seven alleles per population. Among the 14 Glu-V1 alleles, one produced no HMW-glutenin subunits, ten coded for a single subunit, and three for two subunits. The mobilities of all the subunits in SDS-PAGE gels were greater than that of reference subunit 7 of Triticum aestivum cv Chinese Spring. Eight of the alleles were relatively abundant (mean frequency over all populations ranged from 0.08 to 0.17) and distributed widely among the 14 populations (8 to 14); five of the alleles were rare (0.003 to 0.021) and found in only 1 to 5 populations. The frequencies of two alleles could not be individually estimated because of the similar electrophoretic mobility of their subunits. The multiple-allelic diversity at Glu-V1 was high (He ranged from 0.700 to 0.857) but similar from population to population. Overall, about 7% of the total allelic variation was distributed among populations (Gst=0.072), and more than 90% within populations. Whether the allelic variation at Glu-V1 is subject to natural selection is unknown, but the discovery of the homozygous null Glu-V1 alleles in the present study may be useful in pursuing this question. The multiple-allelic diversity in Glu-V1 presents the plant breeder with an opportunity to evaluate and select the most useful alleles for transfer to wheat. The importance of an evaluation genetic diversity in a wild species before interspecific gene transfers are attempted is well illustrated in this study.