Taxonomic position of Otonycteris hemprichii always has been uncertain, although traditionally it has been considered closely related to Nycticeius, Rhogeessa, and Scotophilus in the tribe Nycticeini (Chiroptera: Vespertilionidae). A recent reevaluation of morphologic and karyotypic data alternatively placed Otonycteris in the tribe Plecotini with Barbastella, Corynorhinus, Euderma, Idionycteris, and Plecotus. We assessed taxonomic position of Otonycteris by analyzing a broad taxonomic sample, including nycticeine, plecotine, and antrozoine bats. Parsimony analysis of DNA sequences from about 2.7 kilobases of the mitochondrial genome (12S rRNA, tRNA^Val, 16S rRNA) indicated that Otonycteris is related more closely to Antrozous and Rhogeessa than to any traditional plecotines. The analysis also supported monophyly for traditional plecotines. Not all relationships were resolved within the plecotines, but analysis corroborated previous studies in the sister relationship between Idionycteris and Euderma and monophyly of the Old World genus Plecotus and the New World genus Corynorhinus. Our phylogeny contradicts the most recent assessment of plecotine bats but is congruent with much of the available morphologic and karyotypic data on relationships within and among nycticeine and plecotine bats.