Due to the small area and the cascaded structure formed by surface water, alpine micro-waterbodies are often considered to have similar habitats to alpine streams. However, due to the differences between the environmental factors and the benthic diversity, the functions of these two habitats in the ecosystem may be completely different. Northwest Yunnan hosts one of the richest global biodiversity hotspots where alpine micro-waterbodies and streams are densely concentrated. These two fresh water ecosystems have important functions in regional benthic biodiversity maintenance, however, these peculiar freshwater ecosystems have barely received research attention. In order to compare the similarities and differences of environmental factors and biodiversity between these two habitats and their maintenance effects on benthic diversity, in this study, benthic biodiversity and the environmental factors of 27 alpine micro-waterbodies and a stream in the same region (9 sample sites within an altitude gradient of 500 m) were investigated in an alpine valley of Gongshan County, Nujiang, Yunnan Province in June of 2015. Results showed that: (1) The common characteristics of benthic communities in alpine micro-waterbodies and the stream were that the dominant taxa have large population size, while, the rare taxa had higher richness but small population size. (2) However, the environmental factors, species diversity, functional diversity and community structures were quite different between the alpine micro-waterbodies and the stream, the alpine stream had higher species richness, species diversity and functional diversity than alpine micro-waterbodies. (3) the benthic biodiversity and formation of community structure in alpine stream were related to the elevation and aquatic environmental factors relating to flow rate regulation, while, the aquatic environmental factors of alpine micro-waterbodies did not act as the drivers for the distribution of benthic biodiversity. Therefore, these findings suggest that alpine micro-waterbodies and streams are distinct ecosystems that each feature has very different characteristics, they cannot be regarded as similar ecosystem types. Both of them play an important role in the maintenance of regional benthic biodiversity and ecosystem functions.