Endemic Hawaiian Drosophilidae, a radiation of nearly 1000 species including 13 federally listed as endangered, occur mostly in intact native forest, 500–1500 m above sea level. But their persistence in disturbed forest and agricultural areas has not been documented. Thus, control efforts for agricultural pests may impact endemic species if previously undocumented refugia in agricultural areas may play a role in their conservation. To quantify whether invasive plants and agriculture habitats may harbor endemic Drosophilidae, we established standardized trapping arrays, with traps typically designed to control invasive fruit flies (Tephritidae), with 81 sites across native, disturbed and agricultural land use gradients on the islands of Hawai’i and Maui. We collected and identified, to species level, over 22,000 specimens. We found 121 of the possible 292 species expected to occur in the sampled areas, and the majority (91%) of the captured specimens belonged to 24 common species. Species diversity and numbers were greatest in the native forest, but 55% of the species occurred in the invasive strawberry guava belt and plantation forest, adjacent to and almost 500 m from native forest, and 22 species were collected in orchards and nonnative forest as far as 10 km from native habitats. Their persistence outside of native forest suggests that more careful management of disturbed forest and a reassessment of its conservation value are in order. Conservation efforts and assessments of native forest integrity should include the subset of species restricted to intact native forest, since these species are highly localized and particularly sensitive. Additionally, future efforts to control invasive pest fruit flies should consider the nontarget impacts of maintaining traps in and near native forest. This survey project demonstrates the utility of thorough biotic surveys and taxonomic expertise in developing both sensitive species lists and baseline diversity indices for future conservation and monitoring efforts.