SUMMARY Lint percentage is an important component of cotton yield traits and an important economic indicator of cotton production. The initial stage of fiber development is a critical developmental period that affects the lint percentage trait, but the genetic regulation of the initial stage of fiber development needs to be resolved. In this study, we used a genomewide association study (GWAS) to identify 11 quantitative trait loci (QTLs) related to lint percentage and identified a total of 13 859 expression QTL (eQTLs) through transcriptome sequencing of 312 upland cotton accessions. Candidate genes for improving the lint percentage trait were identified through transcriptome‐wide association study (TWAS), colocalization analysis, and differentially expressed gene analysis. We located nine candidate genes through the TWAS, and prioritized two key candidate genes ( Ghir_A12G025980 and Ghir_A12G025990 ) related to lint percentage through colocalization and differential expression analysis. We showed that two eQTL hotspots (Hot26 and Hot28) synergistically participate in regulating the biological pathways of fiber initiation and development. Additionally, we unlocked the potential of genomic variants in improving the lint percentage by aggregating favorable alleles in accessions. New accessions suitable for improving lint percentage were excavated.