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The glassy-winged sharpshooter, Homalodisca coagulata (Homoptera: Cicadellidae), is a highly polyphagous insect species that is distributed throughout most of the southern regions of the United States. In the last 10 years, H. coagualta has become established in California and represents a significant threat to the state's $35 billion wine and table grape industries. DNA sequencing analysis was used to characterize a portion of the mitochondrial cytochrome oxidase I gene from a single population of the smoke tree sharpshooter, Homalodisca liturata, in California and from 20 natural populations of H. coagulata distributed in Tahiti, California, Texas, Louisiana, Mississippi, Alabama, and Florida. The results indicate that H. liturata and H. coagulata are genetically distinct, suggesting that they do not hybridize. Populations of H. coagulata are geographically structured into two groups of haplotypes; a group of populations from east of the Mississippi River including Louisiana, Mississippi, Alabama and Florida and a group comprised of populations west of the Mississippi River from Texas and California, and from Tahiti. There was no genetic structure among haplotypes within the eastern and western groups, respectively. The data also indicates that H. coagulata in California most likely originated from a source in Texas and not from any of the populations east of the Mississippi River.