The Anteholosticha-Holosticha complex is an extremely divergent group within the urostylids, especially because the genus characterization lacks suitable synapomorphies. Previous studies have shown that morphological classification of species within this group often conflicts with SSU-rDNA data, that is this complex is not recovered as a monophyletic group and Anteholosticha spp. are widely dispersed throughout the urostylid assemblage in SSU-rDNA trees. In this study, we provided 38 new sequences (including the type species of Anteholosticha) of SSU-rDNA, ITS1-5.8S-ITS2 and LSU-rDNA genes to infer molecular phylogenies of all available taxa in the Anteholosticha-Holosticha complex. The results show that: (1) Holosticha is monophyletic in all trees, suggesting it is a well-defined genus; (2) Anteholosticha is polyphyletic and distinctly separated from Holosticha in all single-gene based and concatenated phylogenies; (3) the monophyly of Arcuseries, a recently established genus split from Anteholosticha, is strongly supported by all molecular data; (4) Anteholosticha multicirrata, Anteholosticha manca, Anteholosticha paramanca and Bakuella subtropica may share a most recent common ancestor; (5) multi-gene analyses receive higher support values than the single-gene analyses.