Biogeography research is flawed by the poor understanding of microbial distributions due to the lack of a systematic research framework, especially regarding appropriate study units. By combining pure culture and molecular methods, we studied the biogeographic patterns of nematode-trapping fungi by collecting and analysing 2,250 specimens from 228 sites in Yunnan Province, China. We found typical watershed patterns at the species and genetic levels of nematode-trapping fungi. The results showed that microbial biogeography could be better understood by 1) using watersheds as research units, 2) removing the coverup of widespread species, and 3) applying good sampling efforts and strategies. We suggest that watersheds could help unify the understanding of the biogeographic patterns of animals, plants, and microbes and may also help account for the historical and contemporary factors driving species distributions.