The distribution pattern and maintenance mechanism of biodiversity in ecosystem has always been a core topic in ecological research. Microorganism is the junction of material circulation in all ecosystems on earth, understanding the distribution pattern of microbial diversity has become the key to spatial distribution pattern of the whole biodiversity. However, little is known about the mechanism of the spatial pattern of microbial diversity. One reason is a long-term neglect of the interrelationships between microorganisms in terrestrial and aquatic ecosystems. And considering the strong diffusion capacity and great diversity of microorganisms, it is necessary to systematically set up symmetrical samples on terrestrial and in aquatic in areas with a strong environmental heterogeneity and carry out qualitative and quantitative measurements by combining pure culture and molecular methods. Nematode-trapping fungi (NTF) are eukaryotic microorganisms easy to isolate, purify and identify. Currently, only 106 species of NTF are known, which are widely distributed in various terrestrial and aquatic habitats around the world. It is efficient to use NTF to study the distribution patterns of microorganisms. We investigated the diversity of NTF on terrestrial and in aquatic in the two tributaries of Lancang River, where has high environmental heterogeneity, to explore the differences and interrelations of microorganism distributions between terrestrial and aquatic in watershed. The primary tributaries of the Lancang River, including the Bijiang River and the Heihui River, were selected as the study areas in low-water seasons, and 12 sampling points were set up systematically in confluent areas of two rivers, 5 terrestrial samples and 5 aquatic symmetrical samples were collected at each sampling point, and a total of 120 samples were collected. Combined the traditional methods of isolation and purification with morphological and molecular biological technology, a total of 88 strains, 13 species and 2 genera were obtained according to the Nematode-Trapping Fungi. Among which 45 strains, 11 species and 2 genera were isolated from the terrestrial samples with detection rates of 41.67%, while 43 strains, 10 species and 1 genus were isolated from the aquatic samples with detection rates of 53.33%. The results showed that there were differences between terrestrial and aquatic in species composition, genera and community structure of NTF in the two tributaries of the Lancang River. The terrestrial soil may be the origin of NTF diversity in the watershed, and water flow is an important transmission factor. In watershed ecosystems, the dispersal restriction between terrestrial and aquatic and the connectivity of water flow are important mechanisms for maintaining microbial diversity.