Cotton produces natural fiber for the textile industry. The genetic effects of genomic structural variations underlying agronomic traits remain unclear. Here, we generate two high-quality genomes of *Gossypium hirsutum* cv. NDM8 and *Gossypium barbadense* acc. Pima90, and identify large-scale structural variations in the two species and 1,081 *G. hirsutum* accessions. The density of structural variations is higher in the D-subgenome than in the A-subgenome, indicating that the D-subgenome undergoes stronger selection during species formation and variety development. Many structural variations in genes and/or regulatory regions potentially influencing agronomic traits were discovered. Of 446 significantly associated structural variations, those for fiber quality and *Verticillium* wilt resistance are located mainly in the D-subgenome and those for yield mainly in the A-subgenome. Our research provides insight into the role of structural variations in genotype-to-phenotype relationships and their potential utility in crop improvement.

Zhiying Ma, Yan Zhang, Liqiang Wu, Guiyin Zhang, Zhengwen Sun, Zhikun Li, Yafei Jiang, Huifeng Ke, Bin Chen, Zhengwen Liu, Qishen Gu, Zhicheng Wang, Guoning Wang, Jun Yang, Jinhua Wu, Yuanyuan Yan, Chengsheng Meng, Lihua Li, Xiuxin Li, Shaojing Mo, Nan Wu, Limei Ma, Liting Chen, Man Zhang, Aijun Si, Zhanwu Yang, Nan Wang, Lizhu Wu, Dongmei Zhang, Yanru Cui, Jing Cui, Xing Lv, Yang Li, Rongkang Shi, Yihong Duan, Shilin Tian, Xingfen Wang