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This study was performed to identify marker loci associated with important agronomic traits and oil content under two moisture conditions and find stable associations in test environments in a worldwide collection of safflower (Carthamus tinctorius L.). Association analysis was conducted between eight important traits and 341 polymorphic AFLP markers produced by 10 primer combinations (EcoRI/MseI) in 100 safflower genotypes. The results of population structure analysis identified three main subpopulations possessing significant genetic differences revealed by analysis of molecular variance. Association analysis explained the highest percentage of trait variation for seed yield (38%) under drought-stress conditions and number of seeds per capitulum (27.75%) under normal conditions. Four markers (M51/E41-6, M51/E41-4, M61/E40-6 and M62/E40-17) in drought-stress conditions and two markers (M62/E40-35 and M47/E37-13) in normal conditions were simultaneously associated with seed and oil yield. The markers stably associated with traits in all test environments included M62/E40-35 with seed yield in normal conditions, M62/E40-17 with seed yield in drought stress conditions, and M62/E41-11 with oil yield in drought-stress conditions. Significant relationships were identified between oil content and three markers (M61/E40-6, M47/E37-8 and M51/E32-9) under drought-stress conditions, and three markers (M61/E2-2, M61/E40-6 and M51/E41-12) under normal conditions. Among them, M51/E32-9 and M61/E2-2 markers showed stable association with oil content under drought-stress and normal conditions, respectively. Detected markers would be useful in marker-assisted breeding programs for safflower improvement in arid and semi-arid area.