Background: Data on the current species diversity from the Drosophilidae family in South Africa is limited or outdated.Objectives: Using haphazard, limited trapping for a different study, we serendipitously report on and document Drosophilidae species in two distinct regions (representing a sub-tropical and a Mediterranean climate region) of South Africa.Method: Drosophilidae were trapped using mixed fruit and mushroom traps around urban areas in two climatically distinct regions of South Africa. The flies were identified using standard barcoding (Cytochrome c Oxidase Subunit I [COI] gene sequence) and, in some cases, additional identification from a taxonomical expert using morphological traits. Species were checked against literature, online resources and a previously compiled library of South African Drosophilidae to determine whether they were new records.Results: Thirteen species were readily collected and identified. Of these, three species (Drosophila ananassae, Drosophila nasuta and Zaprionus taronus) have not been reported previously in South Africa. One of the species (Z. taronus) was captured in a home garden, while the other two species were captured in an urban-agricultural region with a sub-tropical climate.Conclusions: From our limited serendipitous sampling, three new species records have been found in sub-tropical climates in South Africa. With more comprehensive, systematic sampling, a better understanding of the South African Drosophilidae composition, and thus the detection of alien or invasive species, can be pursued. Baseline data for understanding spatio-temporal patterns of native biodiversity, or for informing management actions in the case of alien or invasive species, are currently inadequate for this group in the region.