The aphid Therioaphis trifolii (Monell) is indigenous to the western Palaearctic region, where it colonizes lucerne (alfalfa), clovers and related legumes. A form of the species, T. trifolii f. maculata (the spotted alfalfa aphid, SAA), has been known in Australia since 1977, feeding almost exclusively on Medicago sativa (lucerne). Since 1989, T. trifolii has been found in Australia colonizing Trifolium subterraneum (subclover). We have compared samples of T. trifolii in Australia collected on lucerne and subclover using several techniques: survival and reproduction on different hosts, morphology, cuticular hydrocarbon profile, karyology, and a combination of RAPD-PCR and mitochondrial DNA (cytochrome oxidase) genetic markers. Whereas there were no distinct differences in cuticular hydrocarbons and karyology, we found significant correlated differences between the host on which aphids were collected, some morphological characters, and the genotypes of the aphids. Aphids collected from lucerne were always of one genetic type, and nearly all those from subclover were of another. Both groups of aphids are morphologically distinguishable from the yellow clover aphid (YCA), the other form of the species recognized so far. We conclude that the clover-colonizers (spotted clover aphid, SCA) and the lucerne-colonizers (SAA) are each host-restricted forms (biotypes) of T. trifolii. Estimates of mtDNA divergence are in the range of conspecific to closely congeneric, as compared with other insects including aphids. We developed simple diagnostic DNA tests to distinguish the two pest aphids.