Cultivated and wild species of the genus Vigna were screened for their flavonoid content. Flavonoid HPLC analyses clearly showed that cultivated lines of cowpea (Vigna unguiculata L. Walp.) are very similar from a qualitative point of view, always showing three flavonoid aglycons: quercetin, kaempferol, and isorhamnetin. In addition, a positive relationship between resistance/susceptibility characteristics against aphids and flavonoid glycoside content of cowpea lines was found. The resistant lines showed a flavonoid content higher than that of susceptible ones. In vitro bioassays proved that, among endogenous flavonoids, quercetin and isorhamnetin possess a good inhibitory aphid reproduction rate. Flavonoid HPLC analyses of wild Vigna species supported evidence for the existence of different flavonoid chemotypes in some species of section Vigna. There are kaempferol chemotypes, kaempferol being the main aglycon detected, quercetin chemotypes, containing quercetin glycosides only, and two isorhamnetin chemotypes. When the resistance characteristics to aphids in different chemotypes of the same species were tested, it became evident that quercetin or isorhamnetin chemotypes showed a higher level of resistance compared to kaempferol chemotypes in the same species, thus demonstrating a direct involvement of quercetin or isorhamnetin in the resistance mechanism. These results can provide useful information for further studies on gene expression of resistance factors.