The southeastern boll weevil, the Mexican boll weevil, and the thurberia weevil are considered to be morphologically similar but behaviorally different variants of the same species, Anthonomus grandis Boheman. A polymerase chain reaction (PCR)-amplified 9.2-kb section of the mitochondrial DNA was cleaved with restriction enzymes. RFLPs of weevils from three cotton growing locations in Texas and one in northeastern Mexico were compared with thurberia weevil from three sites in Arizona. Six haplotypes were observed in the Texas/Mexico collections and 12 haplotypes were found among the thurberia weevil. There were no shared haplotypes between these two groups. Polymorphism was observed within the weevil types. The three thurberia weevil locations exhibit some geographic isolation and exhibit differences in both the haplotypes present and the relative frequencies of the haplotypes. Only one haplotype was recovered at all three Arizona sites. The Texas/Mexico samples showed less genetic variability with the northern most site having the lowest polymorphism. 52/53 of these weevils appear to be genetically southeastern boll weevil. Two haplotypes were shared by all four of these populations and comprised 72% of the insects examined. The range of genetic distances between haplotypes was <0.001–0.022. The Mexican boll weevil was not explicitly examined; however, three individuals were discovered that appear to represent a genetically distinct third population. One was from Mexico and the other two were from a thurberia weevil site. These three individuals may represent the Mexican boll weevil. The results include apparent diagnostic restriction fragment differences between the thurberia weevil and the southeastern boll weevil that could be used to help determine whether future weevils found in Arizona or California cotton are thurberia weevil, southeastern boll weevil, or another population of weevils.