Higher-level relationships among the earliest lineages of brachyceran Diptera remain poorly resolved by comparative morphology. Nucleotide sequence data should be useful in clarifying brachyceran relationships, especially where morphological evidence is either contradictory or controversial. We examined phylogenetic relationships among the family-level taxa of the brachyceran infraorder Tabanomorpha using sequences of a large portion of the 28S ribosomal DNA. Twenty-five species were sequenced, including five outgroup species from the Stratiomyomorpha and Xylophagomorpha. Parsimony and maximum likelihood-based phylogenetic analysis of 2,371 alignable sites yielded identical inferred tree topologies. 28S rDNA supports the monophyly of the Tabanomorpha (Vermileonidae, Rhagionidae, Pelecorhynchidae, Athericidae and Tabanidae). Our results contradict several published hypotheses that associate Vermileonidae with asiloid or eremoneuran taxa remote from the Tabanomorpha. The molecular data also support monophyly for all of the included family-level lineages, and corroborate several recent phylogenetic hypotheses based on comparative morphology.