Molecular markers are useful to improve germplasm collection management and for identifying genes for future enhancement and breeding. Safflower (Carthamus tinctorius L.) accessions from the U.S. germplasm collection were characterized using AFLP (Amplified Length Polymorphisms) markers. After DNA extraction and PCR reactions, separation and scoring of 392 markers was completed using the Beckman CEQ8000 capillary electrophoresis system. Twelve plants from each of eight populations were analyzed for an estimate of within population similarity. Bulked leaves of eight plants of 93 accessions from seven regions (the Americas, China, E. Africa, E. Europe, the Mediterranean, S.C. Asia, and S.W. Asia) were also analyzed to compare within and among regional molecular variation. The within population similarity for the cultivar Girard at 0.91 was significantly higher than six of the seven other accessions tested indicating generally lower diversity. The Arizona Wild Composite (AWC), with high phenotypic diversity, had a similarity of 0.81, which was significantly lower than all other populations. The results were consistent with the expectation that plants within a cultivar would be generally less diverse, and plants from the AWC more diverse. Within regions, accessions from China had the highest similarity and those from E. Europe and the Americas the lowest similarity; that is, the most diversity. Principal coordinate analysis showed that the Americas overlapped with Europe and E. Africa and to a lesser extent with the Mediterranean and China. However, excluding the Americas and East Europe, the remaining five regions were distinguished by the principal coordinates fairly well, and S.W. Asia was clearly different from all other regions. The results showed that AFLP markers are useful for determining within and among patterns of genetic diversity in safflower germplasm.