Astigmata comprise a diverse group of acariform mite species with a remarkable range of life histories, most of which involve parasitic or commensal relationships with other organisms. Several authors have suggested that Astigmata evolved as a paedomorphic clade from within Oribatida, and both morphology and gland-chemistry strongly suggest that their sister-clade is within the oribatid subgroup Desmonomata. The biologies of these groups contrast greatly, since oribatid mites are mostly soil-living detritivores and fungivores, and have life cycles that are much longer than those in Astigmata. We tested the hypothesis that Astigmata evolved from within Desmonomata using two molecular markers, the ribosomal 18S region (18S) and the nuclear elongation factor 1 alpha (ef1alpha) gene. Representative acariform mites included 28 species of Oribatida, eight of Astigmata, two of Prostigmata and two of Endeostigmata; outgroups included members of Opilioacariformes, Parasitiformes and Ricinulei. To minimize the possibility of long-branch attraction artifacts, we limited highly variable sites by removing gaps (18S) and third codon positions (ef1alpha) from the sequences. Maximum parsimony, neighbor-joining and Bayesian algorithms formed trees that consistently placed Astigmata outside monophyletic Oribatida, usually as sister-group of the endeostigmatid mite Alicorhagia sp. Analyses with and without outgroups resulted in similar topologies, showing no evidence for long-branch artifacts and leaving the conflict with morphological and biochemical data unexplained.