The tribe Dacini (Diptera: Tephritidae: Trypetinae) currently consists of ≈40 genera and 1,000 described species. Among others, it includes the genera Bactrocera, Ceratitis, and Dacus, all of which are pests of agricultural importance because they oviposit in ripening fruit. Although previous studies, based on morphological and molecular data, suggested that the members of Dacini are monophyletic, no extensive molecular phylogenetic studies have been published on Bactrocera, Ceratitis, or Dacus. Therefore, we undertook a phylogenetic analysis of 34 tephritid taxa, including 16 species of the tribe Dacini, utilizing 1,391 bp from the mitochondrial 16S rRNA, 12S rRNA, and cytochrome oxidase II tRNA_Lys tRNA_Asp genes. Combined maximum parsimony analysis suggested the following with strong quantitative support: (1) the subfamilies Tephritinae and Trypetinae are monophyletic, (2) Dacini is monophyletic, and (3) members of the subtribe Dacina are monophyletic. In addition, Ceratitidina Gastrozonina are basal to Dacina. Within Dacina, Dacus and Bactrocera were each monophyletic. The results obtained here are generally congruent with those based on morphology.