Abstract: Biological soil crusts (BSCs) play critical ecological functions in desert ecosystem. Microcoleus sp. are key filamentous cyanobacteria and play vital roles in BSCs. More and more strains of Microcoleaceae were found in desert areas, however, it is difficult to distinguish them in species level due to similar morphological characteristic. In this manuscript, 11 filamentous cyanobacterial strains with similar morphology to Microcoleus were isolated and purified from BSCs in Gurbantonggute Desert. The experimental cyanobacterial strains were examined morphologically as well as phylogenetically using 16S rRNA gene and the 16S—23S internal transcribed spacer (ITS) rigion. The results show that the experimental cyanobacterial strains belong to the genera Microcoleus and Symplocastrum, including two newly recorded species in China: M. steenstrupii and S. flechtnerii, as well as M. vaginatus and a suspect species similar to M. steenstrupii. The number and alignment of cyanobacterial filaments, cell size and the shape of apical cells, and phylogenetic relationship based on 16S rRNA are key evidence to identity different species of Microcoleaceae. Secondary structure of ITS are also vital reference to distinguish to different species in same genus.