The genetic structure of 23 populations of Graphocephala atropunctata (Signoret) (Hemiptera: Cicadellidae: Cicadellinae), a vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., was investigated using ribosomal 28S and mitochondrial cytochrome C oxidase I gene sequences. The 28S sequences were identical across all G. atropunctata specimens and populations, but 16 mitochondrial haplotypes were detected and significant interpopulation differences were found in the distribution of these haplotypes. Pairwise estimates of F(st) correlated positively with geographical distance between populations, a phenomenon known as genetic isolation by distance. Thus, despite potential for widespread movement of G. atropunctata through nursery and agricultural industries, isolated populations of G. atropunctata have remained genetically distinct, suggesting that negligible numbers of G. atropunctata are actually transported or population interbreeding rarely occurs. The phylogenetic relationship between G. atropunctata and two additional congeners, Graphocephala cythura Baker and Graphocephala flavovittata Metcalf, which have overlapping distributions with G. atropunctata, also was investigated. Although 28S sequences of G. flavovittata were strikingly similar to those of G. atropunctata, mitochondrial DNA (mtDNA) suggests that both species are genetically distinct from G. atropunctata.