Bemisia tabaci (Gennadius) represents a relatively large cryptic species complex. Australia has at least two native populations of B. tabaci sensu lato and these were first found on different host plants in different parts of Australia. The species status of these populations has not been resolved, although their mitochondrial sequences differ by 3.82–4.20%. We addressed the question of whether these AUSI and AUSII B. tabaci populations are distinct species. We used reciprocal cross-mating tests to establish whether the insects from these different populations recognize one another as potential mating partners. The results show that the two native Australian populations of B. tabaci have a mating sequence with four phases, each of which is described. Not all pairs in the control crosses mated and the frequency of mating differed across them. Some pairs in the AUSI-M × AUSII-F did mate (15%) and did produce female progeny, but the frequency was extremely low relative to controls. Microsatellite genotyping of the female progeny produced in the crosses showed these matings were successful. None of the AUSII-M × AUSI-F crosses mated although some of the males did search for females. These results demonstrate the critical role of the mate recognition process and the need to assess this directly in cross-mating tests if the species status of different populations is to be tested realistically. In short, AUSI and AUSII B. tabaci populations are distinct species because the individual males and females do not recognize individuals of the alternative population as potential mating partners.