The cotton leafworm, S. littoralis, is a major pest in Egypt causes severe quantitative and qualitative losses of cotton and other economic crops. This study examined the effect of chitin synthesis inhibitors, lufenuron (match 5%) on the 2nd instar larvae. The results showed that lufenuron caused not only mortality in larval stages, but also caused effect on pupal and adult duration, adult fecundity and egg fertility. Also lufenuron treatments have resulted in emergence of deformed pupae and adults. PCR-RFLP technique was used in this study to investigate the effect of lufenuron on the vira-like chitinase gene, one of the genes involved in the formation of chitin and concerned with development of insect. The endonucleases Spel, EcoRI and Bbsl were used to detect the molecular changes at different time intervals 6, 12, 24 and 48hrs post treatment with sublethal doses LC25, LC50 and LC90.The results showed significant changes in the sequences of the treated groups which were dose and time dependants. Moreover the number of RNA secondary structure stems was varied in the different studied groups and most of them differed in their nucleotide sequences, positions and free energy.