Cassava is an important food crop, energy crop and industrial raw material. Cassava mealybug (Phenacoccus manihoti Matile-Ferrero) is a dangerous quarantine pest in the world, the cultivation and utilization of insect-resistant cassava cultivars can effectively block its colonization. Mining insect-resistant secondary metabolites and the regulatory genes is one of the important strategies for insect-resistant breeding. Flavonoids are unique secondary metabolites for plants to resist biotic and abiotic stresses, but the function of flavonoids and the synthesis pathway genes in cassava resistance to cassava mealybug is still unclear. Based on this, this study analyzed the expression levels of flavonoid synthesis pathway-related genes (CHS, CHI, FLS, LAR, DFR, F3H, CCoAOMT, C4H, C3'H and ANR) and flavonoid content in the leaves of insect-resistant (C1115, SC9, Myanmar) and insect-susceptible (KU50, SC205 and Bread) cassava cultivars after being damaged by cassava mealybug for different time (0, 1, 4, 8 d). Although the expression of CCoAOMT, C3'H, ANR and C4H in the leaf was up-regulated after feeding, there was no significant difference compared with that before damage, and there was no significant difference in the expression level between the resistant and susceptible cassava cultivars. In contrast, the expression levels of CHS, CHI, FLS, F3H, DFR and LAR genes were significantly higher than those before damage, and in the same damage time, the expression levels of the six genes in the insect-resistant cassava cultivars were also significantly higher than those in the insect-susceptible cassava cultivars. Further correlation analysis showed that the expression levels of CHS, CHI, FLS, F3H and LAR genes were significantly positively correlated with the insect resistance of cassava cultivars. In addition, the results of total flavonoid content determination showed that the total flavonoid content increased significantly after one day of damage compared with that before damage, and the total flavonoid content of insect-resistant cassava cultivars was significantly higher than that of the insect-susceptible cassava cultivars after 4 days of damage. Correlation analysis showed that the total flavonoid content was also significantly positively correlated with the insect resistance of cassava cultivars. Therefore, it is speculated that the increase of flavonoid content and the significantly up-regulation of CHS, CHI, FLS, F3H and LAR in the insect-resistant cassava cultivars may be related to the resistance to cassava mealybug. This study would provide an important preliminary basis for the in-depth analysis of the molecular mechanism of flavonoid synthesis genes regulating the insect-resistant defense response of cassava to mealybug, as well as the molecular design and breeding of cassava insect-resistant.