Key message <p><b>The high fiber strength (FS) of a Upland cotton cultivar bred by interspecific cross was found to be associated with a D11 QTL and the identification of a candidate gene, and a genetic variant provides a potential tool for molecular breeding of high FS cotton cultivars.</b></p> Abstract <p>Fiber strength (FS) is one of the critical determinants of cotton fiber quality. Despite many quantitative trait loci (QTLs) associated with FS have been documented, many more are yet to be uncovered. In this study, an F₂ population was constructed by crossing Huiyuan720 (<i>Gossypium hirsutum</i>) with Shida26-22, a high FS <i>G. hirsutum</i> cultivar bred by introgressing the high FS trait of <i>G. barbadense</i> cultivar Xinhai53 into Huiyuan720 via multiple backcrosses. Bulked segregant analysis sequencing of the F₂ population identified six FS QTLs, including a major locus on chromosome D11 spanning 3.38&#xa0;Mb containing 121 annotated genes. By integrating transcriptome sequencing of developing fibers, analyzing gene co-expression network, and profiling genetic variants of annotated genes, <i>GH_D11G2131</i>, annotated to encode an armadillo repeat/tetratricopeptide repeat-like protein, was identified as a strong candidate gene potentially regulating FS. <i>GH_D11G2131</i> harbored a non‑synonymous mutation and a promoter indel between the two parents, belonged to a co‑expression gene module highly correlated with cell wall development, and was significantly highly expressed in 20&#xa0;days post‑anthesis fibers of high FS cultivars. Virus-induced gene silencing demonstrated that knockdown of <i>GH_D11G2131</i> resulted in thinner secondary cell walls and a significant reduction in lignin content, thanks to the marked downregulation of genes related to lignin biosynthesis. Collectively, this study uncovered a major FS QTL and a strong candidate gene associated with SCW development, providing a potential molecular tool for breeding elite cotton cultivars with high fiber quality.</p>