Cottonseed contains high quality protein meal for feed and oil for human consumption, but gossypol in cottonseed has potential toxicity and detrimental effects that limit cottonseed use as food for humans and monogastric animals. Therefore, identifying germplasm containing lower gossypol content is critical. The objective of this research was to investigate the influence of specific chromosomes or chromosome segments from Gossypium barbadense, G. tomentosum, and G. mustelinum, respectively, on (+) and (−) gossypol levels when substituted into G. hirsutum. A total of 11 genotypes were used in this study: nine chromosome substitution lines (CS lines) were investigated for cottonseed gossypol level in field experiments in 2013 and 2014; TM-1 (the recurrent parent of the CS line) and AM UA48 (cultivar) were used as controls. Results showed significant variation in gossypol level and its fractions among CS lines. This variation is a result of chromosome substitution, although it was also affected by environment (location) as location x genotype was significant. Significant positive relationships between total gossypol, (+) gossypol, and (−) gossypol were found. This research demonstrated significant differences among the nine CS lines, and some CS lines had significantly lower gossypol level in cottonseed. These results provide an alternative breeding approach for possibly selecting low levels of gossypol and improving cottonseed nutritional qualities using CS lines.