We analyzed the phylogenetic relationships of the genusTrisetacususing two genes [cytochromecoxidase subunit I (COI) and D1-D2 region of 28S rDNA (D1-D2 28S)], a representive taxon sampling (nearly 40% of known diversity), and a large set of close and distant outgroups. Our analyses suggest the presence of a dichotomy betweenTrisetacusassociated with Cupressaceae and Pinaceae. The following smaller molecular clades were found:Pin-1(bud mites, twig sheath mites, bark gall mites, and endoparasitic mites from pinaceans),Pin-2(needle sheath mites from pines),Pin-2a(putative Nearctic group of needle sheath mites),Pin-2b(putative Palearctic group of needle sheath mites),Cup-1and2(bud, cone, seed mites and mites living under bark scales from cupressaceans). The monophyly of the recently proposed subgenusBrevithecusnested within cladeCup-2was confirmed. Ancestral character reconstruction analyses recovered: (1) Pinaceae as the ancestral hosts of Nalepellidae andTrisetacus, (2) repetitive reductions of the spermathecal tube independently occurred in two lineages ofTrisetacusfrom Cupressaceae, and (3) several mite habitats on host (galls, cones, twig sheaths, seeds, inside leaves, and under scales) are evolutionarily derived states, whereas living in buds or needle sheaths are ancestral states forTrisetacuscladesCupandPin. Using confocal microscopy, we identified six basic types of the female internal genitalia ofTrisetacusbased on shapes of the spermatheca and spermathecal tube. These genitalic types are strongly correlated with lineages recovered by molecular phylogenetic analyses, suggesting that the female genital morphology is both evolutionarily conserved and is a factor influencing macroevolutionary patterns in this group of mites.