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Two progeny populations of upland cotton derived from mass selection-mass crossing, MSF and its family lines MSF, were generated from 17 hybrid cotton lines derived from regional trials conducted in the Yellow River basin and Yangtze River basin in China. These populations were used to verify 39 reported molecular markers that were related to quantitative trait loci (QTLs) for Verticillium wilt resistance of cotton. Only 12 of 39 markers were polymorphic; 19 had no polymorphisms, and amplification failed for eight markers. The differences in disease grades of aa/AA genotype individuals for five markers, BNL3241, NAU1225, NAU1230, JESPR153, and BNL3031, reached either significant or highly significant levels in at least one population. These markers can thus be effectively used for marker-assisted selection (MAS) of the target trait. Especially for JESPR153 and BNL3031, the differences in disease grades of aa/AA genotype individuals both reached either significant or highly significant levels in the two populations. These two markers should be given preferential consideration when undertaking MAS. The two flanking markers were more effective than the single flanking marker for MAS of single-loci QTL. The selection effect will be greatly enhanced through a reasonable allocation of marker combinations for multi-locus QTL polymerization. When using multi-locus markers for multi-locus QTL-assisted polymerization breeding, the selection effect can be improved progressively by increasing the number of polymerization markers. The possible interaction of different QTLs or genetic backgrounds does not influence the selection effect. A combination of resistant genotypes and disease grade performance enabled final selection of three individuals resistant to Verticillium wilt. [ABSTRACT FROM AUTHOR]