We genetically characterized Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) populations from Texas, Florida, and Mexico and the sister taxon Tamarixia triozae (Burks) by two molecular methods. T. radiata is an ectoparasitoid of Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), nymphs. The populations were submitted to intersimple sequence repeat-polymerase chain reaction DNA fingerprinting with two primers. No fixed banding pattern differences were uncovered among the populations of T. radiata with either primer, whereas different patterns were observed in T. triozae, suggesting that there is no genetic differentiation among the populations. Support for these results was obtained by sequence analyses of the internal transcribed spacer region one and the mitochondrial cytochrome oxidase subunit one (COI) gene. In both genes, the intrapopulational variation range (percentage divergence, %D) fell within the interpopulational variation range. The %D at the COI gene between T. radiata and T. triozae was 9.0–10.3%. However, haplotype structure was uncovered among the populations. No haplotypes were shared between Florida and Texas/Mexico, whereas sharing was observed between Texas and Mexico. Two population-specific nucleotides were identified that allowed the discrimination of the Florida and Texas/ Mexico individuals. A neighbor-joining and a parsimonious tree clustered the populations into two distinct clades. The Florida population clustered into one clade, whereas the Texas/Mexico populations clustered into another clade. The COI phylogeographic analysis suggests that the population of T. radiata in Texas did not come from Florida. The data also suggest that the population in Texas came from Mexico, or vice versa, because the Mexico population showed less haplotype diversity.