This investigation aimed to detect the pesticide residues in cotton seeds and effects of these pesticide residues on fatty acids of the organic cotton seeds Giza 86 variety and treated cotton seeds Giza 86 variety harvested from the experimental plots and cultivated during the seasons 2017 and 2018. Gas chromatography (GC) with flame photometry (FPD) was used in determining residual pesticides and changes in fatty acids. Fatty acids were determined using GC/Mass spectrometry. Results indicated the existence of detectable residues of the insecticide Chlorpyriphos in treated cottonseeds Giza 86 variety (conventional samples) in both seasons 2017 and 2018 at the range of 1.11 ppm, and 1.14 ppm for Chlorpyriphos, and 6.51 ppm, and 5.30 ppm for Profenophos. The resulting values were hazardous and toxic according to Maximum residue levels (MRLs). The results revealed an increasing value of Myristic acid in the organic Giza 86 at 2.74% than the treated variety at 1.13% and evanescence of the Pentadecanoic acid (C15:0) in the inorganic variety. Palmitoleic acid (C16:1 w9), Hexadecatrienoic acid (C16:3w4), linoleic acid (C18:3 w3), Arachidic acid (C18:3 w3), and Docosenoic acid (C22:1 w 11) appeared in the treated variety Giza 86 with disappearance in the organic variety. Decreasing the value of Stearic acid (C18:0) from 6.34% in the organic Giza 86 to 3.91% in the treated variety. Vaccinic acid (C18:1 w7) was decreased from 1.68% (organic Giza 86) to 0.93% (treated Giza 86), and oleic acid (C18:1w9) from 31.14% (organic Giza 86) to 24.26% (treated Giza 86). Contrariwise, the fatty acid Linoleic acid C18:2w6 increased from 31.45% in the organic Giza 86 to 42.20% in the treated Giza 86 and also slightly increased the value of Palmitic acid (C16:0) at 24.39% in the treated variety Giza 86 than 24.14% in the organic variety.