Wild barley [Hordeum vulgare ssp. spontaneum (C. Koch) Thell.] is a part of the primary gene pool with valuable sources of beneficial genes for barley improvement. This study attempted to develop a core subset of 269 accessions representing 16 countries from the Plant Gene Resources of Canada (PGRC) collection of 3,782 accessions, and to characterize them using barley simple sequence repeat (SSR) markers. Twenty-five informative primer pairs were applied to screen all samples and 359 alleles were detected over seven barley chromosomes. Analyses of the SSR data showed the effectiveness of the stratified sampling applied in capturing country-wise SSR variation. The frequencies of polymorphic alleles ranged from 0.004 to 0.708 and averaged 0.072. More than 24% or 7% SSR variation resided among accessions of 16 countries or two regions, respectively. Accessions from Israel and Jordan were genetically most diverse, while accessions from Lebanon and Greece were most differentiated. Four and five optimal clusters of accessions were obtained using STRUCTURE and BAPS programs and partitioned 16.3% and 20.3% SSR variations, respectively. The five optimal clusters varied in size from 15 to 104 and two clusters had only country-specific accessions. A genetic separation was detected between the accessions east and west of the Zagros Mountains only at the country, not the individual, level. These SSR patterns enhance our understanding of the wild barley gene pool, and are significant for conserving wild barley germplasm and exploring new sources of useful genes for barley improvement.