The cereal rust mite Abacarus hystrix (Nalepa), a significant pest of grasses, has been regarded as one of a few exceptions among eriophyoid mites with reference to the pattern of host plant utilization. At least 60 grass species have been recorded as its hosts. Thus, the mite has long been considered as a host generalist in which host specialization would not be likely to evolve. However, recent studies have revealed that host-associated specialization is possible in A. hystrix. Here, we aimed to discriminate between the three populations of A. hystrix associated with the different hosts (namely quackgrass, ryegrass and smooth brome) on the basis of mitochondrial (COI) and nuclear (D2 region of 28S rDNA) DNA sequences. The phylogenetic trees obtained with the maximum likelihood analysis of both COI and D2 region data sets showed that host-adapted strains of A. hystrix form distinct clades. Furthermore, on the COI nucleotide tree, the quackgrass- and brome-associated strains were internally divided each into two well-supported monophyletic clusters. The nucleotide D2 region data set tree showed that brome-associated strain is polyphyletic in origin. There is clear co-variation of DNA results with earlier morphological and ecological traits, as well as the results of crossing experiments. We showed that reproductively incompatible strains of A. hystrix exhibit more than 20% sequence divergence in the COI gene and 0.2% sequence divergence in the D2 28S rDNA. Our results did not confirm the placement of three host-associated strains of A. hystrix within one, ostensibly generalist, species.