We analyzed the ecological-genomic diversity of microsatellites of wild barley, Hordeum spontaneum (C. Koch) Thell., at 18 loci in 306 individuals of 16 populations from Jordan across a southward transect of increasing aridity. The 18 microsatellites revealed a total of 249 alleles, with an average of 13.8 alleles per locus (range 3-29), with nonrandom distribution. The proportion of polymorphic loci per population averaged 0.91 (range 0.83-1.00); gene diversity, He, averaged 0.512 (range 0.38-0.651). We compared the number of alleles of the 18 loci to those found in Israel populations by Turpeinen et al. Out of the 280 alleles, 138 (49.3%) were unique (i.e. occurred in only one of the countries). The percentage of unique alleles in Jordan and Israel populations was 43.0% and 17.9%, respectively, suggesting that Jordan is an important center of origin and diversity of wild barley. Estimates of mean gene diversity were highest in the populations collected near the Golan Heights, such as Shuni North, Shuni South and Jarash. Sixty nine percent of the microsatellite variation was partitioned within populations and 31% between populations. Associations between ecogeographical values and gene diversity were established for eight microsatellite loci. The cluster produced by simple sequence repeat (SSR) data is mostly coincidence with the result of the dendrogram of the Spalax ehrenbergi superspecies of subterranean mole rats in Jordan based on allozyme gene loci. The major soil type in the wild barley habitat of each ecological group was different. Stepwise multiple regression analysis indicated that the variance of gene diversity was explained by altitude (R(2) = 0.362**). These observations suggest that microsatellites are at least partly adaptive and subject to natural selection.