The simultaneous improvement of fiber strength (FS) and lint percentage (LP) is a critical objective for achieving high-quality and high-yield cotton production. Identifying key genes and their regulatory networks that govern the synergistic development of FS and LP is essential for achieving their simultaneous improvement. In our previous study, a stable chromosome segment, Seg-D06-2, was identified for its ability to concurrently enhance both FS and LP with high reliability. In the present study, homozygous individuals harboring the Seg-D06-2 segment within a nearly uniform genetic background were selected to construct a large BC₆F₂ chromosome segment substitution line (CSSL) population comprising 3324 individuals. Extreme individuals characterized by simultaneous improvement in FS and LP, which shared similar genetic and phenotypic backgrounds, were subjected to comparative transcriptomic and weighted gene co-expression network analysis (WGCNA) at 0, 5, 10, 15, 20, and 25 days post-anthesis (DPA). The results highlighted the ’blue’ and ’yellow’ modules as being significantly associated with the simultaneous improvement of FS and LP. Four hub genes (GH_D06G0542, GH_D06G1609, GH_D06G0627 and GH_D06G2689) and two DEGs (GH_D06G0564 and GH_D06G0723) were identified in the ’blue’ module. Three hub genes (GH_D06G0540, GH_D06G0558 and GH_D06G0636) and one DEG (GH_D06G0527) were identified in the ’yellow’ module. These 10 key genes likely play pivotal roles in regulating the synergistic development of FS and LP, warranting further investigation. The reliability of the RNA-seq data was confirmed by qRT-PCR. This study provides a valuable resource for molecular breeding aimed at the simultaneous improvement of FS and LP and offers new insights into the molecular mechanisms governing their synergistic development.