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The thief ant, Solenopsis molesta (Say), a common nuisance ant species found throughout the United States is genetically related to other stinging ants. This research was undertaken to estimate genetic variation utilizing mitochondrial DNA sequences of cytochrome oxidase I in thief ant populations from various geographic locations. Thief ants were collected from nine states, identified to species using morphologic characteristics from available keys. DNA extractions were completed with Qiagen’s Gentra PUREGENE® DNA Isolation Kit using their solid tissue protocol. PCR reactions were run on the extracted DNA using primers Lep-F1 (forward) and Lep-R1 (reverse). The resulting DNA products were concentrated and purified by Microcon Centrifugal Filter Unit YM-100. Purified samples were sent to University of Arkansas Medical Sciences (UAMS) DNA Sequencing Core Facility for sequencing. Sequences obtained from UAMS were edited and align using Codon Code Aligner. The contigs were then uploaded to www.Phylogeny.fr and a phylogenetic tree was produced. The tree obtained displayed variation in genetic makeup of the thief ants collected and the genetic variation corresponded to the morphologic identification. Using several phylogenetic tree rendering software programs, (Neighbor joining, Maximum likelihood and Bayesian) found at www.Phylogeny.fr, the thief ants collected from the different states were separated into three groups. Ants collected from New York, Indiana and one location in Nebraska forming one group identified as S. molesta validiuscula and another with ants from Louisiana identified as S. carolinensis and the third group comprised of ants from, South Dakota, Washington, New Jersey, Tennessee, Kansas and two other locations in Nebraska identified as S. molesta molesta. DNA barcodes were generated from 600bp sequences of the cytochrome c oxidase subunit 1 (COI) and are available for use as a molecular diagnostic tool for identification for these three species of thief ants. COI sequences of thief ants collected from the nine states were submitted to GenBank and were issued accession numbers HM179641 to HM179653.