Seasonal, spatial, and habitat responses of carrion-associated Diptera assemblages can provide valuable information about the presence or absence of species and their relative abundance, and thereby enhance understanding of their responses to environmental variables and how this may have an impact on forensic investigations. Three different nature reserves (localities) within the Municipality of Tshwane, South Africa, were selected to determine whether species assemblages of carrion-feeding flies differ between seasons, localities, and habitat types. A total of 59,511 adult Diptera, identified to 35 species in eight different families, were collected using modified Redtop hanging traps, baited with liver and fish, during four seasons in three different habitat types. Species assemblages differed temporally, with season being the main factor determining species diversity and not locality or habitat. However, savanna and human-disturbed habitats supported a higher abundance and species richness than grassland habitats. Areas adjacent to the localities, such as large urban expanses in Dinokeng or agricultural holdings in Rietvlei, led to an increase in the abundance and mean species richness of carrion-associated Diptera, and in increased numbers of pest or invasive species such as Chrysomya megacephala (F.). Despite this, the overall species assemblages present in human-disturbed areas were very similar to those recorded in natural habitats.