DNA sequence data were used to examine phylogenetic relationships between six species of economically important Tetranychidae mites: Eotetranychus carpini (Oudemans), E. pruni (Reck), Tetranychus pacificus McGregor, T. mcdanieli McGregor, T. turkestani Ugarov & Nikolski and T. urticae Koch. With primers directed toward conserved elements flanking the target region, the Polymerase Chain Reaction was used to amplify the ITS2 spacer of the ribosomal DNA molecule. The nucleotide sequence of a 300-bp fragment of the ITS2 was determined by direct sequencing and nucleotide divergence used for intra-generic comparison in mites. The resulting phylogenetic tree expressing interspecific relationships in genus Tetranychus agrees with morphological data. The study demonstrates the usefulness of the approach in the assessment of the systematics and evolution of the group.