AbstractTetranychid spider mites are a significant worldwide pest of agriculture with Tetranychus urticae being the most important. In Australian cotton, T. urticae is the most damaging, but Tetranychus ludeni and Tetranychus lambi are also present and can be difficult to distinguish morphologically. For this reason, a fast‐diagnostic assay was developed to identify T. urticae, T. lambi and T. ludeni in a single real‐time PCR reaction. The assay design was based on a universal primer pair to amplify the internal transcribed spacer of nuclear ribosomal DNA (ITS1) with three species‐specific TaqMan probes. The assay was validated by sequencing three known reference strains for T. urticae, T. lambi and T. ludeni and a blind test on 14 DNA samples that included species other than the three target species. The method correctly identified the target species and produced a negative result against other non‐target species. Furthermore, we examined 22 field‐collected spider mite populations from Australian cotton that had been laboratory maintained. Here, we found that 50% of the populations labelled as T. urticae were mixed with T. lambi and quarter of the populations labelled with T. lambi contained T. urticae. It demonstrates just how difficult it is to maintain strain integrity with multiple species in proximity. Finally, the assay was used by Biosecurity Australia to test 2125 mite quarantine intercept specimens. They found 1949 T. urticae, 1 T. lambi, 13 T. ludeni and 162 unknown specimens confirming assay accuracy and robustness. The diagnostic assay developed can provide quick spider mite species identification and can support multiple uses including species identification to support spray decisions or the precise identification of border quarantine intercepts where accuracy and turnaround time is critical.