Cotton serves both as fiber crop and source of plant protein and oil. Simultaneous improvement of seed nutrition and fiber quality is critical to raise cotton economic benefit, yet the genetic basis of cottonseed protein content and its relationship with fiber strength remain unclear. We developed high-precision NIRS prediction models for cottonseed protein and oil using 628 upland cotton accessions. GWAS identified GhMYB86 as a pleiotropic gene modulating cottonseed protein content and fiber strength. Functional KASP markers were developed for molecular-assisted selection. This study provides valuable gene resources and detection tools for cotton quality breeding.