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Insect cuticular hydrocarbons (CHCs) have dual functions as physical barrier and chemical signals. The last step of CHC biosynthesis is known to be catalyzed by cytochrome P450 CYP4G in a number of insects. Until recently, studies on CYP4Gs in the context of functional evolution are rare. In this study, we analyzed sequence similarity and temporal-spatial expression patterns of the five CYP4G genes in the cotton bollworm Helicoverpa armigera , an important agricultural pest and also typical representative of lepidopteran insects. Moreover, the CRISPR/Cas9-induced knockout was used to clarify the roles of the five CYP4Gs in CHC biosynthesis. Temporal-spatial expression patterns revealed that CYP4G8 was highly expressed at all developmental stages and in most tissues examined. Larvae with CYP4G8 knocked out could not produce methyl-branched CHCs and failed to pupate, while larvae with the other four CYP4G genes knocked out (4G1-type-KO) showed no significant changes in their CHC profiles, weight gain and survival. Comparative transcriptomics revealed that knocking out CYP4G8 affected the global gene expression in larvae, especially down-regulated the expression of genes in the fatty acid biosynthetic pathway, while no significant change in 4G1-type-KO transcriptome was observed. These findings indicate that the five members of the CYP4G subfamily have undergone functional divergence: CYP4G8 maintains the essential function in CHC biosynthesis, while the function of the other four CYP4G genes remains unclear. Intriguingly, CYP4G8 has evolved to be a P450 enzyme responsible for the synthesis of larval methyl-branched hydrocarbons. The observation that CYP4G8 knockout is lethal strongly suggest that CYP4G8 may serve as a candidate target for the development of insecticidal agents for the control of cotton bollworms. [Display omitted] • CRISPR/Cas9 was used to characterize the function of five CYP4G genes from Helicoverpa armigera. • CYP4G paralogs diverge in spatio-temporal expression and function. • CYP4G8, rather than the other four CYP4Gs, is the major player in cuticular hydrocarbon biosynthesis. • CYP4G8 knockout is lethal. [ABSTRACT FROM AUTHOR]