The invasive red imported fire ant, Solenopsis invicta Buren, has successfully dispersed across many countries from its South American homeland and now has reached the US-Mexico border (e.g., Matamoros, state of Tamaulipas, México), where it now coexists with native fire ants, Solenopsis geminata, Solenopsis xyloni, and others. The morphological identification of Solenopsis spp. workers is difficult, particularly small ones. We examined the sequence of the cytochrome oxidase I (COI) mitochondrial gene (mtDNA) as a marker for fire ants collected at several Mexican localities. PCR products from this locus yielded unique sequences and restriction patterns that allowed distinguishing between S. invicta, S. geminata, and specimens harboring S. xyloni sequences. The S. invicta sequences obtained were 99% identical to sequences reported from Florida and New Mexico specimens. The S. xyloni sequences obtained were 96% identical to New Mexico sequences. The S. geminata sequences were similar (93% identity) to those from Florida, and shared a Hinf I restriction site with some but not all Florida sequences. The S. xyloni sequences were detected in S. geminata/S. xyloni hybrids identified by morphology; along with other characters, the marker allows their characterization.