Abstract: Flavonoids are important chemicals of resistance to pests in cotton plant. The main flavonoid chemicals and their spatio temporal dynamics of content in Bt transgenic cotton were tested by HPLC. The results showed that the flavonoid chemicals of resistance to pests mainly including rutin, isoquercitrin and quercetin could be detected and quantitatively analyzed by HPLC. The contents of rutin, isoquercitrin and quercetin were the highest in petal, but lower in calyx, bract and cotton boll. Moreover, the total content of flavonoid chemicals in top leaf was much higher at developmental stage than at seedling stage. The content of each flavonoid chemicals of resistance to pests was different during different developmental stage and in different organs. It was indicated that different flavonoid chemicals played different roles in resistance to pests.