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For millennia, wheat (Triticum spp.) has been grown in traditional aflaj-irrigation systems of remote mountain oases in Oman. However, little is known about the diversity of the ancient landraces used. Given recent reports about the occurrence of novel germplasm in such material, the objective of this study was to evaluate the genetic diversity of hexaploid wheat (Triticum aestivum L.) landraces in relation to their geographic origin using microsatellites. The collection covered most of the cultivation areas in northern Oman where wheat landraces are growing. Total genomic DNA was extracted from six pooled plants representing each accession. A total of 161 wheat accessions were assayed using 35 microsatellite loci in which a total of 305 polymorphic bands were recorded for the 35 microsatellites. The polymorphic information content (PIC) across the 35 microsatellite loci ranged from 0.02 to 0.89 with an average of 0.50. A heterozygosity percentage value of 9.09 was determined and the highest level recorded for accessions from the Batinah district. Rare alleles averaged 1.85 with the highest value being from the Dakhilia district. The results indicated a significant correlation between gene diversity and number of alleles across districts. The correlation coefficient between these two variables over the 35 loci was 0.657, whereby correlation coefficients of 0.718, 0.706, 0.657 and 0.651, respectively, were found for the Batinah, Dhahira, Dakhilia and Sharqia materials. Genetic distances indicated that all landraces were closely related. The cluster analysis discriminated most of the landraces accessions. However, it failed to achieve region-specific groupings of landraces. The present study demonstrated the presence of high diversity in Omani landraces and also indicated the effectiveness of microsatellites to describe it.