The Astigmata, a large and variable group, is still a subject of taxonomic dispute. Particularly, their origin from ancestors of the lower oribatid mites (e.g., Malaconothroidea) seems well documented by many lines of evidence. The structure of spermatozoa has been successfully applied to phylogenetic investigations in many animal groups. The aim of our study was to provide new data on spermatozoon structure in Astigmata and to consider its appropriateness in phylogenetic studies. The study reveals information on spermatozoa in 17 species of Astigmata (11 species studied for the first time) extending our knowledge to 18 species (one species known only from the literature) representing 12 families and 7 superfamilies. Spermatozoa have the same basic structure in all species: cells are multiform and the chromatin forms thin threads embedded directly in the cytoplasm; the acrosome is absent. The cytoplasm in most species contains electron-dense lamellae, varying in both number and arrangement within the cell. In Sarcoptoidea, electron-dense tubules in contact with lamellae margins were also observed in Psoroptidae (Psoroptes equi), whereas in two representatives of Sarcoptidae (Notoedres cati and Sarcoptes scabiei), only electron-dense tubules were found. In two species, Canestrinia sellnicki (Canestrinioidea: Canestriniidae) and Scutulanyssus obscurus (Analgoidea: Pteronyssidae), neither lamellae nor tubules were present. The mitochondria in a spermatozoon are usually gathered at the cell periphery and their structure is usually modified to form so-called mitochondrial derivatives. The chromatin threads are an autapomorphy strongly supporting the monophyly of Astigmata. As spermatozoa vary considerably between species in Astigmata, we deduce that sperm structure may be useful for phylogenetic analyses within the group. Several conclusions concerning the affinities within Astigmata are presented. Spermatology seems to be unhelpful, however, in questions on the origin of Astigmata (particularly for Astigmata–Oribatida relationships), since their sperm do not possess synapomorphies with sperm of the remaining groups of Acariformes, i.e., Endeostigmata, Prostigmata, and Oribatida. J. Morphol. © 2005 Wiley-Liss, Inc.