Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the progenitor of cultivated emmer wheat (ssp. dicoccum) and durum wheat (ssp. turgidum conv. durum). Because of its full interfertility with domesticated emmer wheat, this wild species can serve as one of the most important genetic resources to improve durum as well as bread wheat. To elucidate the magnitude of genetic variation within a population of wild emmer wheat, variation of chloroplast DNA was investigated using 91 plants, in total, collected from two natural habitats in southern Turkey. Allelic variation at 24 microsatellite loci in the chloroplast genome was investigated using these samples. Allelic variations were observed at 15 microsatellite loci. The number of alleles per locus was the same in the two populations, ranging from 1 to 4 with an average of 2.17. The estimated diversity indices (H) were also very close ranging from 0.00 to 0.70 with an average of 0.28 and 0.29 for the two populations. Based on the observed allelic variation at all chloroplast microsatellite loci, a total of 23 chloroplast haplotypes (plastotypes) were identified. Only two plastotypes were shared in common between the two natural populations, indicating that the two populations are highly differentiated. Furthermore, uneven micro-geographic distribution of plastotypes was found within each population, suggesting limited rate of migration (seeds dispersal rate) in this species. Our study demonstrated the presence of a high level of genetic diversity between and within highly structured populations of wild emmer wheat in southern Turkey.