In an effort to improve surveillance capacity for the exotic red imported fire ant, Solenopsis invicta, a lateral flow immunoassay (LFA) was recently evaluated by Biosecurity Queensland staff in Australia. The purpose of the research was to assess the ability of the fire ant LFA to discriminate S. invicta from ants found in Australia and to conduct the first field evaluation of the test. In addition to S. invicta, 36 species of ants, collected mainly from Queensland, were evaluated by the LFA, including species from the Dolichoderinae (n = 7), Formicinae (n = 13), Myrmeciinae (n = 1), Myrmicinae (n = 11), Ponerinae (n = 3) and Pseudomyrmicinae (n = 1) subfamilies. The fire ant LFA test correctly identified S. invicta in every instance. No cross reactivity was observed in the other ant species. Field tests by staff previously unfamiliar with the test resulted in suggestions for improving ant collection and manipulation. The fire ant LFA appears to be suitable for use in Australia for rapid confirmation of potential new detections of S. invicta.