China is the most species-rich region for the Unionoidea (Unionidae + Margaritiferidae) in East Asia, with a total of 26 genera. However, in the context of global unionids, an integrated and scientific phylogenetic taxonomy for Chinese Unionoidea is still lack. Based on the multilocus molecular markers (COI, 16S, 28S) and mitochondrial genomics, we reconstructed the phylogenetic relationship of the Unionioda by focusing on the Unionoidea. All phylogenetic results of ML, BI and BEAST supported five monophyletic clades for subfamily levels within Unionidae, i.e. (((Ambleminae + Gonideinae) + Rectidentinae) + Unioninae) + Parreysiinae, and two subfamilies within Margaritiferidae, i.e. (Gibbosulinae + Margaritiferinae). Chinese Unionoidea belonged to four subfamilies (Gonideinae, Rectidentinae, Unioninae, Parreysiinae) thirteen tribes of Unionidae and 2 subfamilies (Gibbosulinae, Margaritiferinae) of Margaritiferidae. The fossil-calibrated molecular clock suggested the Unionidae species divergence at the Middle Triassic (median=232.07 Ma). The origin and species divergence time of the Parreysiinae were earlier than other subfamilies. The species divergence time of Rectidentinae was the latest among all subfamilies, at the Late Cretaceous (median=94.72 Ma). The most recently common ancestor of Ambleminae and Gonideinae diverged at the Late Jurassic (median=144.69 Ma). Margaritiferidae originated at the Late Carboniferous (median=312.61 Ma), and species divergence at the Late Cretaceous (median=91.59 Ma). The oldest living unionid species in China could be traced back to the Cretaceous (i.e. Acuticosta chinensis, median=114.36 Ma). Mitochondrial gene order (GO) analysis of the Unionoidea revealed that the presently published Unionoidea mitochondrial genome had four gene arrangements:one was exclusive to the Margaritiferidae (GO1); Unioninae and Ambleminae of Unionidae shared another one (GO2); Gonideinae had two gene arrangements (GO3 and GO4), of which was exclusive to Chamberlainia hainesiana (GO4). The morphological features of shells, glochidia and marsupium, which were used to high-level classification, could not be regarded as diagnostic features for the subfamily and the tribe level. This study reconstructed the phylogenetic relationship and evolutionary history of Unionoida worldwide and established complete systematics of Unionoidea in China, which providing the taxonomic basis for the diversity research and resource conservation of Chinese unionids.