The present study was conducted to assess the molecular diversity among four populations of spiny bollworm from different insecticides treated Egyptian governorates (Gharbia, Kafr ELShaikh, Daqahlia and Behira) and a population from untreated field cotton. The protein profile displayed deviation in the resulted bands among the studied samples. The distribution of peroxidase and polyphenol oxidase isoenzymes and the intensity of peroxidase presented no difference between the tested samples when compared to control population and each other. While, there was a variation in the intensity of the detected polyphenol oxidase isoenzyme. Data of RAPD-PCR presented a variation in DNA bands of the studied populations. As well as, the similarity index revealed that the control population recorded low similarity index with the other studied populations. Furthermore, it the studied population could be clustered into 2 groups Daqahlia and Behira (100% similar) and Gharbia and Kafr El Shaikh (92%). Therefore, it could be concluded that Earias insulana larvae were adapted to the environment changes and the use of insecticides.