Genome size estimates for both sexes of forensically relevant Diptera from 17 species (four families) are reported herein. Average genome sizes ranged from 425.8 Mb for female Chrysomya rufifacies to 1,197.4 Mb for male Haematobia irritans. These estimates are useful not only for molecular studies, but also for determination of the species and sex of immatures. Species in three of the sampled families had sexually dimorphic genome sizes, presenting a new tool useful for the determination of sex in these species, especially in the immature stages where sexes are morphologically difficult or impossible to identify. In addition, closely related species had significantly different genome sizes, suggesting the use of flow cytometry as a new tool for species identification of some species of forensically relevant larvae.