Safflower is an important oilseed crop mainly grown in the arid and semi-arid regions of the world. The aim of this study was to explore phenotypic and genetic diversity, population structure, and marker-trait association for 100-seed weight in 94 safflower accessions originating from 26 countries using silicoDArT markers. Analysis of variance revealed statistically significant genotypic effects (p< 0.01), while Turkey samples resulted in higher 100-seed weight compared to Pakistan samples. A Constellation plot divided the studied germplasm into two populations on the basis of their 100-seed weight. Various mean genetic diversity parameters including observed number of alleles (1.99), effective number of alleles (1.54), Shannon's information index (0.48), expected heterozygosity (0.32), and unbiased expected heterozygosity (0.32) for the entire population exhibited sufficient genetic diversity using 12232 silicoDArT markers. Analysis of molecular variance (AMOVA) revealed that most of the variations (91%) in world safflower panel are due to differences within country groups. A model-based structure grouped the 94 safflower accessions into populations A, B, C and an admixture population upon membership coefficient. Neighbor joining analysis grouped the safflower accessions into two populations (A and B). Principal coordinate analysis (PCoA) also clustered the safflower accessions on the basis of geographical origin. Three accessions; Egypt-5, Egypt-2, and India-2 revealed the highest genetic distance and hence might be recommended as candidate parental lines for safflower breeding programs. The mixed linear model i.e., the Q + K model, demonstrated that two DArTseq markers (DArT-45483051 and DArT-15672391) had significant association (p< 0.01) for 100-seed weight. We envisage that identified DArTseq markers associated with 100-seed weight will be helpful to develop high-yielding cultivars of safflower through marker-assisted breeding in the near future.