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Background  The  Anopheles hyrcanus  group includes 25 species, and is widely distributed in the Oriental and Palaearctic regions. Several species within this group are vectors of malaria, lymphatic filariasis and Japanese encephalitis. It is difficult or impossible to identify cryptic species based on their morphological characteristics, with some closely related species of the Hyrcanus Group have similar adult morphological characteristics. Thus, their molecular identification has been an important complementary method to traditional morphological taxonomy.    Methods  We used 461 ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) sequences relating to 19 species to reconstruct the molecular phylogeny of the Hyrcanus Group across its range. In addition, we compared the performance of rDNA ITS2 to that of mitochondrial DNA (mtDNA) cytochrome  c  oxidase subunit 1 gene ( cox 1) to assess the genetic divergence of Hyrcanus Group sibling species.    Results  Based on Kimura’s 2-parameter (K2P) distance model, the average conspecific ITS2 divergence was 0.003, whereas sequence divergence between species averaged 0.480. Average ITS2 sequence divergences were almost 160 times higher among the Hyrcanus Group members than within each species. Two sets of sibling species,  An. lesteri  Baisas & Hu, 1936 and  An. paraliae  Sandosham, 1959; and  An. sinensis  Wiedemann, 1828,  An. belenrae  Rueda, 2005, and  An. kleini  Rueda, 2005, were resolved by ITS2. Each of these species was represented as an independent lineage in the phylogenetic tree. Results suggest that  An. pseudopictus  Grassi, 1899 and  An. hyrcanus  (Pallas, 1771) are most likely a single species. We uncovered two new ITS2 lineages that require further study before resolving their true taxonomic status, and designed a diagnostic polymerase chain reaction (PCR) assay to distinguish five morphologically similar species.    Conclusions  Nuclear and mitochondrial genes generally provided consistent results for subgroup division. Compared to  cox 1, ITS2 is a more reliable tool for studying phylogenetic relationships among closely related mosquito taxa. Based on species-specific differences in ITS2 sequences, the multiplex PCR assay developed here can be used to improve the efficiency of vector identification. Thus, this research will promote the progress of malaria vector surveillance in both epidemic and non-epidemic areas of South and East Asia.    Electronic supplementary material  The online version of this article (10.1186/s13071-017-2351-x) contains supplementary material, which is available to authorized users.

Molecular phylogeny of Anopheles hyrcanus group members based on ITS2 rDNA