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Grain isozyme and ribosomal DNA (rDNA) variability was examined in Hordeum spontaneum populations sampled from 27 geographical sites in Israel. Considerable phenotypic variability was observed with variants of ADH1, EST3, EST10, BMY1 and WSP detected, which are not available in the H. vulgare gene pool. Seven new rDNA phenotypes were detected in the H. spontaneum populations. Shannon’s index of diversity was used to partition the total phenotypic variation into between and within population components. Most of the variation occurred between H. spontaneum populations. The distribution of both grain isozyme and rDNA phenotvpes was non-random and correlated with a range of ecogeographical factors. In particular, the G phenotype of BMY1 was restricted to the Negev Desert and Dead Sea regions of Israel. Over 78% of the variation in the frequency of this particular phenotype could be explained by the number of rainy days per year and mean temperature in January. This suggests that variation at this locus or at loci linked to it may be of adaptive significance and of value in the introgression of genes controlling abiotic stress tolerance from H. spontaneum into the H. vulgare gene pool.