Phylogenetic trees of spider mites were previously obtained using 18S and 28S rRNA genes. Because some of the bootstrap values were relatively low, these trees were unable to completely resolve the phylogeny. Here, we obtained RNA-Seq data for the 72 known species (73 strains) of spider mites to analyze the phylogeny of the sub-family Tetranychinae. The data were de novo assembled into a total alignment length of 790,047 bases corresponding to 264,133 amino acid residues in 652 genes. The sequence dataset was 200 times larger than the data used in the previous study. The new trees were much more robust and more clearly defined the clades of the tribes and the genera of the sub-family Tetranychinae. The tribe Tetranychini was polyphyletic because a monophyletic clade of Eurytetranychini was placed inside it. The six genera from which two or more species were sampled appeared to be monophyletic, but four genera (Schizotetranychus, Eotetranychus, Oligonychus and Tetranychus) appeared to be polyphyletic. These results strongly support the previous molecular inference of the polyphyletic tribes and genera, although the molecular phylogeny of the sub-family Tetranychinae does not fully agree with the current morphology-based taxonomy. The taxonomy of the sub-family Tetranychinae should be revised according to the molecular relationships revealed by this study.