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Cottonseeds from two parents (TM-1 and 3-79) and their 17 progeny (chromosomal substitution) lines were analyzed for various secondary structures of proteins and moisture content of lipids, separately in hulls and kernels. Fourier transform infrared spectroscopy (FTIR) was used on mature seeds from Upland cotton (G. hirsutum) progeny lines and parents. Based on secondary structures of proteins and hydration levels of lipids, differences were observed among the cottonseeds. The two progeny lines – CS-B12sh and CS-B22sh retained lipid moisture content and protein secondary structures similar to both parents, while CS-B06, CS-B15sh and CS-B16 remained distinct from either parent. On the other hand, CS-B05sh, CS-B07 and CS-B26lo were alike to TM-1 parent for lipid and protein profiles, whereas CS-B02 and CS-B04 were comparable with 3-79 parent. The capacity to detect hydrated and dehydrated lipids and different protein secondary structures using FTIR in these cottonseeds is the novel finding of this project for improving the seed nutritional traits in cotton towards cooking-oil and protein-feed usages.