Phytoplankton has been used as a paradigm for studies of coexistence of species since the publication of the "paradox of the plankton." Although there are a wealth of studies about phytoplankton assemblages of lakes, reservoirs and rivers, our knowledge about phytoplankton biodiversity and its underlying mechanisms in mountain headwater stream ecosystems is limited, especially across regional scales with broad environmental gradients. In this study, we collected 144 phytoplankton samples from the Xijiang headwater streams of the Pearl River across low altitude (< 1,000 m) located in Guangxi province, intermediate altitude (1,000 m < altitude <2,000 m) in Guizhou province and high altitude (> 2,000 m) in Yunnan province of China. Our study revealed high phytoplankton diversity in these streams. Freshwater phytoplankton, including cyanobacteria, Bacillariophyta, Chlorophyta, Rhodophyta, Chrysophyta, Euglenophyta, Glaucophyta, Phaeophyta and Cryptophyta, were all detected. However, phytoplankton alpha diversity exhibited a monotonic decreasing relationship with increasing altitude. High altitudes amplified the "isolated island" effect of headwater streams on phytoplankton assemblages, which were characterized by lower homogeneous selection and higher dispersal limitation. Variability and network vulnerability of phytoplankton assemblages increased with increasing altitudes. Our findings demonstrated diversity, variability and co-occurrence patterns of phytoplankton assemblages linked to environmental factors co-varying with altitude across regional scales.