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Background   Culex  ( Culex )  tritaeniorhynchus  is an important vector of Japanese encephalitis virus (JEV) affecting feral pigs, native mammals and humans. The mosquito species is widely distributed throughout Southeast Asia, Africa and Europe, and thought to be absent in Australia.    Methods  In February and May, 2020 the Medical Entomology unit of the Northern Territory (NT) Top End Health Service collected  Cx. tritaeniorhynchus  female specimens ( n  = 19) from the Darwin and Katherine regions. Specimens were preliminarily identified morphologically as the Vishnui subgroup in subgenus  Culex . Molecular identification was performed using cytochrome  c  oxidase subunit 1 (COI) barcoding, including sequence percentage identity using BLAST and tree-based identification using maximum likelihood analysis in the IQ-TREE software package. Once identified using COI, specimens were reanalysed for diagnostic morphological characters to inform a new taxonomic key to related species from the NT.    Results  Sequence percentage analysis of COI revealed that specimens from the NT shared 99.7% nucleotide identity to a haplotype of  Cx. tritaeniorhynchus  from Dili, Timor-Leste. The phylogenetic analysis showed that the NT specimens formed a monophyletic clade with other  Cx. tritaeniorhynchus  from Southeast Asia and the Middle East. We provide COI barcodes for most NT species from the Vishnui subgroup to aid future identifications, including the first genetic sequences for  Culex  ( Culex )  crinicauda  and the undescribed species  Culex  ( Culex ) sp. No. 32 of Marks. Useful diagnostic morphological characters were identified and are presented in a taxonomic key to adult females to separate  Cx. tritaeniorhynchus  from other members of the Vishnui subgroup from the NT.    Conclusions  We report the detection of  Cx. tritaeniorhynchus  in Australia from the Darwin and Katherine regions of the NT. The vector is likely to be already established in northern Australia, given the wide geographical spread throughout the Top End of the NT. The establishment of  Cx. tritaeniorhynchus  in Australia is a concern to health officials as the species is an important vector of JEV and is now the sixth species from the subgenus  Culex  capable of vectoring JEV in Australia. We suggest that the species must now be continuously monitored during routine mosquito surveillance programmes to determine its current geographical spread and prevent the potential transmission of exotic JEV throughout Australia.    Graphical abstractSupplementary Information  The online version contains supplementary material available at 10.1186/s13071-021-04911-2.

Detection of the Japanese encephalitis vector mosquito Culex tritaeniorhynchus in Australia using molecular diagnostics and morphology