In 2005, a widespread infestation of Aedes albopictus was discovered in the Torres Strait, the region between northern Australia and New Guinea. To contain this species, an eradication program was implemented in 2006. However, the progress of this program is impeded by the difficulty of morphologically separating Ae. albopictus larvae from the endemic species Aedes scutellaris. In this study, three real-time TaqMan polymerase chain reaction assays that target the ribosomal internal transcribed spacer 1 region were developed to rapidly identify Aedes aegypti, Ae. albopictus, and Ae. scutellaris from northern Australia. Individual eggs, larvae, pupae, and adults, as well as the species composition of mixed pools were accurately identified. The assay method was validated using 703 field-collected specimens from the Torres Strait.