Analyses of genetic variation in crop gene pools are a powerful tool for investigating the origin and early evolution of crop lineages. Such analyses also have the potential to identify unique genetic resources for continued crop improvement. The oilseed crop safflower (Carthamus tinctorius) is believed to have been domesticated in the Fertile Crescent region, but up to 10 geographic centers of similarity throughout the world have been proposed based on morphology. Nuclear microsatellite analysis of accessions from each of the 10 proposed centers of similarity, as well as individuals of the progenitor species, suggested the presence of five genetic clusters (1, Europe; 2, Turkey-Iran-Iraq-Afghanistan; 3, Israel-Jordan-Syria; 4, Egypt-Ethiopia; and 5, the Far East-India-Pakistan). North American accessions, products of a secondary introduction from the native range, suggest that a subset of the native accessions harbor unique genetic diversity that could be useful in future breeding efforts. Overall, a Near Eastern origin of safflower was confirmed based on the genetic similarity between the progenitor and the Near Eastern safflower accessions, as well as previous archaeological finds. Genetic differentiation between geographical clusters of accessions is evident, although not to the degree proposed based on morphology.