Optimal exploitation of crop genetic resources requires a knowledge of the range and structure of the variation present in the gene pool of interest. Avena sterilis L., the cultivated oat progenitor, contains a store of genetic diversity that is readily accessible to the oat breeder. The objectives of the present paper were: (1) to evaluate isozyme polymorphisms in a sample of A. sterilis accessions from the U.S. National Small Grains Collection, (2) to analyze the distribution of isozyme diversity across the geographic range of the accessions, (3) to classify the accessions into groups based on isozyme variation, and (4) to suggest strategies for efficient sampling of this germplasm collection. One thousand and five accessions from 23 countries and 679 collection sites were screened for variation using 23 enzyme systems. Due to limited information about the genetic relationship among individual members of families of isozymes in hexaploid oat species, data were recorded solely for band presence. The frequencies of bands in accessions from the various countries were used to calculate the probability of genotypic identity (Ix.y), the probability of a unique genotype (Ux.y), and an adjusted polymorphic index (Hx). Accessions from Turkey and Lebanon had the largest polymorphic index values, Turkish and Moroccan accessions displayed the greatest numbers of bands. Accessions from Iran, Turkey, Iraq, and Lebanon had the largest mean probabilities of containing unique genotypes. Based on isozyme data, Turkey appeared to represent the center of diversity in this germplasm collection. Band frequencies calculated among countries were used in a principal component analysis. Accessions from Israel and Morocco clustered together; accessions from Iran, Iraq, Turkey, and Ethiopia formed another group; and Algerian accessions formed an outlying group. Several isozyme bands had a regional distribution. These results suggested that choosing accessions from countries based on their groupings in the principal component analysis should secure a greater range of diversity than sampling from the collection at random. Cluster analyses based on Jaccard's distances calculated for all pairwise combinations of the 1005 accessions revealed six broad genetic groups of accessions. Groups 1 and 6 contained accessions from many countries and encompassed half of all accessions. Groups 2 and 4 were heavily populated by accessions from Israel and Morocco. Groups 3 and 5 were composed almost exclusively of accessions from Iran, Iraq, and Turkey. By selecting representative accessions from these six groups, oat breeders could most effectively sample the range of genetic variation in this A. sterilis collection.