Plant genomes are not randomly positioned in the nucleus but are packed into higher-order chromatin structures that have important functional implications. In cotton, we assemble reference-grade genome sequences and are interested to know how the linear DNA sequences are packed, the evolutionary process of 3D genome structures between closely related species and their regulatory roles in gene transcription. With the application of Hi-C and related techniques, we obtained the following observations: 1) Genomic structural variations (SVs) preferred occurring in topologically associating domain (TAD) interior instead of TAD boundary and had a large effect on disrupting TAD organization following polyploidization, which led to expression difference of orthologous genes. 2) Active transposon amplification was associated with the increase of the proportion of A compartment in gene regions and had a role in the formation of lineage-specific TAD boundaries. 3) In fiber development, subgenome-relayed switching of the chromatin compartment from active to inactive is coupled with the silencing of developmentally repressed genes, and dissolution of intricate TAD-like structure cliques showing long-range interactions represents a prominent characteristic at the later developmental stage. Dynamic chromatin loops are found to mediate the rewiring of gene regulatory networks that exhibit a significant difference between the At and Dt subgenomes, implicating expression bias of homoeologous genes. These results highlight the necessity of 3D genome study for understanding the reorganization of higher-order chromatin structure and addressing transcriptional regulation evolution underlying agronomic traits in crops.