Water mites of the Hygrobates fluviatilis complex are common in Palaearctic inhabiting mainly running and slowly flowing waters. In this study, we examined the specimens collected in a stream in Golestan Province of Iran which is currently the easternmost distribution limit of the latter species complex. We used a fragment of the mtDNA COI gene and the D2 region of 28S rDNA to test the genetic structure. Molecular analyses revealed presence of two new well-supported species with a mean genetic divergence of 12.80% and 14.66% COI, respectively, from their closest relatives. Phylogenetic analysis based on COI and nuclear DNA data placed H. grabowskii sp. nov. as the sister species of H. persicus, and H. ulii sp. nov. as the sister species of H. turcicus. Discriminant statistical analysis revealed that the two newly discovered species could be separated on the basis of morphometric characters, but robust morphological differences were not found. Yet we were able to show that diversity in water mites is underestimated and that species delimitation requires an integrative approach.