Gossypium barbadense possesses a superior fiber quality because of its fiber length and strength. An in-depth analysis of the underlying genetic mechanism could aid in filling the gap in research regarding fiber strength and could provide helpful information for Gossypium barbadense breeding. Three quantitative trait loci related to fiber strength were identified from a Gossypium barbadense recombinant inbred line (PimaS-7 × 5917) for further analysis. RNA sequencing was performed in the fiber tissues of PimaS-7 × 5917 0–35 days postanthesis. Four specific modules closely related to the secondary wall-thickening stage were obtained using the weighted gene coexpression network analysis. In total, 55 genes were identified as differentially expressed from 4 specific modules. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used for enrichment analysis, and Gbar_D11G032910, Gbar_D08G020540, Gbar_D08G013370, Gbar_D11G033670, and Gbar_D11G029020 were found to regulate fiber strength by playing a role in the composition of structural constituents of cytoskeleton and microtubules during fiber development. Quantitative real-time PCR results confirmed the accuracy of the transcriptome data. This study provides a quick strategy for exploring candidate genes and provides new insights for improving fiber strength in cotton.