The existence of sibling species in the Old World screwworm fly Chrysomya bezziana would raise serious problems in eradicating this pest if it entered Australia. Cytogenetic variation in C. bezziana was investigated by analyzing pupal trichogen polytene chromosomes. Natural populations of C. bezziana spanning its range from southern Africa to Papua New Guinea were examined as well as hybrids between a New Guinea laboratory strain and natural populations. No evidence of sibling species was found. All populations exhibited the same basic banding pattern as the standard sequence established from a Papua New Guinea strain. Extensive asynapsis of chromosome homologues was found in some hybrid crosses and was therefore measured in all populations and hybrids to detect systematic variation. Asynapsis levels in most hybrids could not be statistically distinguished from those present in the parent populations except for crosses between populations at the ends of the range. This result does not permit asynapsis levels to be used in establishing the origin of introduced flies by estimating their distance from known populations. One inversion polymorphism and six band polymorphisms spread over three chromosomes were analyzed. Populations in each sampled region had characteristic combinations of band polymorphisms. This may offer a diagnostic method for determining the origin of flies accidentally introduced to Australia.