he Comet assay, called the single cell gel electrophoresis (SCGE) assay or microgel electrophoresis (MGE) assay, primarily measures DNA strand breakage in single cells according to the cell type in vitro and in vivo experiments. The main objective of this study is to assess the sensitivity of genotoxic to be considered in cotton leafworm, Spodoptera littoralis (Boisd.) larvae by causing depletion in cells responsible for functions of the resistance system of this pest to the pesticides as affected by three compounds; Bacillus thuringiensis, Kurs. (Bactericide); Metarhizium anisopltae, Metsch. (Fungicide) and chitosan (biopolymer) exposed to gamma irradiation at doses of 15, 30 and 60 Gy to increase the purpose of its activity on genotoxicity parameters of the cotton leaf worm, S. littoralis (Boisd.) treated as 4th instars larvae by LC50 ,s of compounds mentioned. B. thuringiensis exposed to gamma doses of 15, 30 & 60 Gy had potentiating efficacy on S. littoralis than B. thuringiensis used alone without exposing to gamma doses. While, M. anisopltae and chitosan had nearest results among tested compounds singly and the same compound exposed to gamma doses used. Different comet assay parameters; Cell numbers, comet length (μ), head diameter (μ), % DNA in the head, tail length (μ), % DNA in tail and % tail moment were affected by direct effect of compounds used on S. littoralis larvae lead to death the most of cells and it's DNA degradation or another effect by division increasing in DNA lead to increase the malformation cells or %DNA as well as examined nuclei numbers compared to normal S. littoralis larvae. Also, % DNA in examined nuclei had destruction grades according to the treatments used that ranged from 0-3 grades. In addition, the resistance system cells of S. littoralis that investigated by fluorescence microscopy had clearly injurious appeared in disruption; lost its roundness and cell wall; swelling or shrinking in the most investigated cells. It can be concluded that chitosan exposed to different gamma doses was the most genotoxicity compound affect on resistance system cells of S. littoralis larvae, followed by B. thuringiensis and M. anisopltae exposed to gamma doses.