Actinidia is taxonomically difficult and economically important. Four traditional barcoding markers, internal transcribed spacer (ITS), rbcL, matK and trnH-psbA, were used to identify the 29 Actinidia species sampled. High-quality sequences could be obtained easily for rbcL, matK and trnH-psbA, and matK performed best at resolving species among these three markers. ITS had a moderate
sequencing success of 72% and the species resolution proportion was 60.7%. Sequencing success rate for matK + rbcL was 97.4% and it discriminated 48.3% of the species analysed. The barcode trnH-psbA could only identify Actinidia eriantha. MatK + rbcL and ITS are useful markers to barcode Actinidia; the utility of ITS in barcoding needs further investigation using high-throughput sequencing technology. Phylogenetic analyses based on ITS, matK, matK + rbcL and matK + rbcL + trnH-psbA indicated Sect. Leiocarpae to be paraphyletic, while Sect. Maculatae and Sect. Strigosae together with Sect. Stellatae formed a monophyletic group. We recommended the subdivision of Actinidia into two groups: one consisting of Sect. Leiocarpae (ovaries glabrous, fruits spotless), and the other comprising sections Maculatae, Strigosae and Stellatae (ovaries hairy, fruits spotted). This study supported the separation of Actinidia chinensis var. chinensis and var. deliciosa at the infraspecific level, and the separation of Actinidia tetramera and Actinidia kolomikta at the specific level. The treatment of Actinidia maloides as a synonym of A. kolomikta and Actinidia cinerascens as a variety of Actinidia fulvicoma was also warranted.