A genetic system which directly measures percent hybridity/heterozygosity, as well as parental line purity, in commercial hybrid cotton (Gossypium hirsutum L.) breeding and seed production is exigent. Variability among morphological and biochemical markers in elite breeding lines and cultivars are lacking. The utility of molecular techniques, here RFLP analysis, to provide desirable qualitative genetic markers was investigated. Initially, 10 different parental lines with known combining abilities were intermated to generate 8 F₁ populations. F₂ populations from 2 parental combinations were also produced. 50 plants were sampled from each parental line, F₁, and F₂ for RFLP analyses. A set of 7-14 potentially useful cDNA probes were selected for each F₁ combination, and at least 2 codominant polymorphic probes per combination were identified for use in further analyses. 2 F₁ combinations consisting of 100%- and 50%-hybrid subpopulations and the 2 F₂ populations produced from the 100%-hybrid F₁'s, were used to test the genetic integrity of the chosen probes. DNA banding patterns from the F₁ subpopulations precisely reflected the respective 100% or 50% genetic contribution of the male parent. Chi-square analyses of the corresponding F₂ populations demonstrated no significant deviation from the expected 1:2:1 genotypic ratio. Known parental line variability and contamination was also discerned and further quantified. Overall results indicate that RFLP's in cotton readily provide an array of simple genetic markers. Furthermore, purity (homogeneity and/or homozygosity) of parental lines per se can be examined and possibly used to maximize heterozygosity and thus heterosis in cotton hybrids.