Apocynum venetum is a crop species with important medicinal value and ecological application potential. However, its breeding and commercial marketing were strictly limited due to the lack of systematic research on its biological characteristics. In order to investigate the geographic distribution of Apocynum venetum resources in the Ili River Valley, and to evaluate the variations on flavonoid content among germplasms and their in vitro antioxidant activity, the germplasm resources in six districts and counties along the Valley were surveyed, and their antioxidant activity of their total flavonoids were evaluated. Apocynum venetum resources were found at 12 sites, with an altitude of 605 to 1376 m. The germplasms showed 75 to 220 cm on plant height, with stem total flavonoid content of 15.92 to 42.31 mg/g (DW), and leaf total flavonoid content of 34.33 to 56.06 mg/g (DW). The total flavonoid content in leaves was higher than that in the stems, while total flavonoid content showed no correlation with either the collection site altitude or plant height (p<0.05). Especially, the samples collected from Apocynum venetum resources in Qapqal Xibe Autonomous County showed that the total flavonoids scavenged 92.97% and 97.40% of DPPH and ABTS radicals with IC50 values of 0.077 mg/mL and 0.143 mg/mL, respectively, and the total antioxidant capacity of T-AOC reached 10.48 μmol/mL. As a result, this study reported the rich genetic diversity on flavonoid content of Apocynum venetum resources in the Ili River Valley, suggesting a potential of their germplasm on genetic improvement and commercial marketing.