Ficus, with about 755 species, diverse habits and complicated co-evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH-psbA, psbK-psbI, atpF-atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra- and inter-specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK-psbI and trnH-psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF-atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH-psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.