Sharon goatgrass (Aegilops sharonensis Eig) is a useful source of genes for wheat (Triticum aestivum L.) improvement, but little is known about its genetic diversity in Israel. We analyzed genetic diversity and population structure of 106 Ae. sharonensis accessions. Twenty-one microsatellites revealed a high level of diversity that was comparable to Triticeae species with larger geographic distribution. The genetic structure of Ae. sharonensis is characterized by high diversity within location and by a short distance (15 km) similarity between populations. Accessions from the Sharon Plain exhibited the highest level of diversity. Because the Sharon Plain is located in the center of geographic distribution of the species and includes the three most genetically diverse sites, it may be the center of diversity of Ae. sharonensis. A wide range of variation was observed in the 11 morphological traits evaluated, and the regional effect on the phenotypic variability was higher than the one observed for genetic diversity. Considerable variability was found in response to leaf rust and stem rust among regions, as a very low frequency of resistance was found for both diseases in the northern regions of Israel. The diversity pattern found in populations of Ae. sharonensis indicate that, to optimize the capture of the greatest diversity, collection and utilization programs should include multiple individuals from as many sites as possible.