Eotetranychus carpini (Oudemans) is an important pest of grapevine (Vitis vinifera L.) in southern Europe. This mite is also found on a number of different plants, including Carpinus betulus L., which commonly occurs in stands and hedgerows bordering vineyards, where it may serve as a potential mite reservoir. The economic importance of this pest has motivated a number of studies aimed at investigating whether the mites found on V. vinifera and C. betulus are conspecific. The results obtained to date have been inconclusive. In this study, we used biological and molecular approaches to investigate this issue. First, we conducted host-switch experiments to test the ability of E. carpini to develop on an alternative host plant, using mite populations originally collected on either C. betulus or V. vinifera plants from the same area. Second, we investigated DNA-based differentiation using nucleotide sequences of the ITS1-5.8S-ITS2 region of the ribosomal DNA of individual E. carpini from the populations examined in our host-plant experiments. We also analyzed sequences of individuals collected in other regions (Italy and Slovenia) to estimate species variation. The results from our host-switch experiments suggest the differentiation of mites collected on the two hosts. Mites collected from C. betulus did not survive and reproduce on V. vinifera and vice versa. Our molecular work revealed significant genetic differentiation between the mites collected from the two hosts, but no evidence of genetic variation among specimens collected from the same host species. Our results indicate the existence of host races of E. carpini.