Abstract.Restriction fragment length polymorphisms in polymerase chain reaction amplified fragments (PCR‐RFLP) of mitochondrial DNA were used to differentiate species of New World screwworms (Diptera: Calliphoridae). Twenty‐seven restriction enzymes were screened on five regions of mtDNA. Eleven restriction fragment length patterns differentiated New World screwworm,Cochliomyia hominivorax(Coquerel), from secondary screwworm,Cochliomyia macellaria(R). Five restriction fragment length patterns were polymorphic inC. hominivoraxwhile all fragment patterns were fixed inC. macellaria.Diagnostic restriction fragment length patterns were used for species diagnosis, whereas intraspecific variable patterns were used to characterize field samples and laboratory strains. The PCR‐RFLP technique is flexible with regard to developmental stage of the sample and method of preservation. We were able to characterize specimens of all life stages from egg to adult including larvae preserved in alcohol and pinned adults. PCR‐RFLP is rapid and inexpensive, enabling specimens to be characterized within 24 h for less th