Key message <p>Two cultivated safflowers from distinct areas elucidate the genetic diversity present in the linoleic acid biosynthesis, flowering time and flavonoid biosynthesis.</p> Abstract <p>The process of domestication facilitates the adaptation of crops to agricultural environments. In this study, we selected two representative safflower cultivars that has been domesticated in two distinct areas in China as samples to investigate their genetic diversity due to local environmental adaption. Yunhong-7 is a locally bred safflower (<i>Carthamus tinctorius</i>) cultivar, that has been currently widely cultivated in Yunnan Province, Southwest China, and Anhui-1 is a safflower cultivar that was locally bred in Anhui Province, East China. We firstly generated the chromosome-scale genome assembly for yunhong-7 cultivar by combining PacBio and Hi-C technologies. Through comparative genomic analysis, we identified structural variations (SVs) between yunhong-7 and anhui-1, which revealed their genetic differences in the pathways of fatty acid biosynthesis, circadian rhythm and flavonoid biosynthesis. Subsequently, a total of 40 non-redundant fatty acid desaturase 2 (FAD2) genes (39 for yunhong-7 and 20 for anhui-1) were identified, revealing the presence of copy-number variation and major genes change between yunhong-7 and anhui-1. The presented results suggested that changes in SVs may induce alterations in the expression of flowering-related genes, which could explain the observed early flowering phenotype in yunhong-7 compared to anhui-1. We identified a total of 197 non-redundant UDP-glucuronosyltransferases <b>(</b>UGT) genes. Based on prokaryotic expression system, we investigated the catalytic functions of two unique UGT genes (<i>CtUGT.18</i> and <i>CtUGT.191</i>). The current study increases our knowledge of genetic diversity among crop cultivars resulting from distinct domestication processes and thus could contribute to the advancement of traits research and the safflower breeding.</p>