The invention discloses a molecular marker closely linked with major QTL sites qBW-chr04-2, qBW-chr05-5, qBW-chr06-3, qBW-chr21-4 and qBW-chr24-1 of the single boll weight of upland cotton. Accordingto the method, a upland cotton intraspecific recombinant inbred line group constructed by hybridizing Shandong cotton research 28*Xinluozao 24 is utilized, and phenotypic evaluation and identificationare carried out on the cotton yield main constituent factor-the single boll weight of the recombinant inbred line group in a multi-year multi-point environment of a northwest inland cotton area and aYellow River basin cotton area. The genotyping and genetic linkage map construction are carried out on the population by utilizing a cotton SNP80K chip. The major sites influencing the single boll weight of cotton and SNP molecular markers closely linked with the sites are obtained through QTL positioning. In the cotton breeding process, the molecular markers are used for auxiliary selection, thesingle boll weight of a new upland cotton variety can be effectively improved in a targeted mode, and therefore the high-yield and high-quality breeding efficiency is improved.