The Deltocephalus-like leafhoppers are a group of grass-feeding insects that share common forms in the male genitalia, chiefly a fused aedeagal-connective structure. The group contains 27 New World genera. The phylogenetic relationships among these genera and other genera in the tribe are unclear because fusion of the aedeagus and connective, although clearly apomorphic, has occurred in both related and unrelated groups. An independent set of characters is required to test the hypothesis that the Deltocephalus-like genera are monophyletic and to derive relationships among these genera. We examined 562 sites in the 3'-end of the mitochondrial 16S ribosomal DNA from 21 species representing 19 New World genera. Eight species in closely related groups or tribes were also analyzed. The region was sequenced directly from PCR-amplified DNA of field-collected and museum-preserved dried specimens. The transversion rate was twice as high as the transition rate; however, 74% of these were adenine-thymine transversions. Analysis of secondary structure indicated that substitution rates were equal in stem and loop forming regions of the processed rRNA. Using either parsimony or distance analysis, derived phylogenies suggested that, with the exception of the genus Cabrulus, the Deltocephalus-like leafhoppers are genera within a monophyletic group.