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Morphologically, early immature stages of the economically important pest called screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), and non-pest secondary screwworms, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), are nearly indistinguishable. Correct identification is crucial to the ongoing eradication and exclusion program protecting the United States, Mexico and Central America from reinvasion of screwworms persistent in South America and the Caribbean. Amplified fragment length polymorphism (AFLP) polymerase chain reaction was used to differentiate populations of C. hominivorax and to discriminate them from C. macellaria. Ten primer pairs screened for interspecific discrimination of C. hominivorax from C. macellaria showed 52 discrete bands, allowing the two species to be readily distinguished; divergent branches on resulting dendrograms showed 100% bootstrap support. C. macellaria populations grouped at the 92% level; C. hominivorax populations grouped at the 68% level. Of the 52 bands, seven were monomorphic for both species, 22 were specific to C. macellaria, ten were present only in C. hominivorax and the remaining 13 bands differentiated C. hominivorax populations. Separate studies using ten strains of C. hominivorax showed a higher level of genetic similarity within than between populations. Analyses using 72 bands (19 monomorphic bands, 53 bands grouped all ten strains at the 58% similarity level) resolved seven mutant strains from Mexico (85% similarity level); all ten strains were resolved at the 72% similarity level. Diagnostic bands were identified for species and strain identification. We conclude that AFLP can be a valuable tool for studies of interspecific and intraspecific genetic variation in screwworm populations.