Abstract: In this study, wild samples of Nostoc flagelliforme were collected across five geographical units, namely Inner Mongolia, Ningxia, Qinghai, Gansu, and Xinjiang. A polyphasic taxonomic and molecular diversity investigation was conducted by integrating morphological observation, 16S rRNA gene sequence analysis, and 16S—23S ITS secondary structure analysis. Phylogenetic trees based on the 16S rRNA gene revealed that N. flagelliforme did not form an independent, highly supported monophyletic clade, but was scattered across multiple evolutionary lineages. 16S rRNA gene similarity among different samples ranged from 98.4% to 99.7%. However, significant intraspecific differentiation was observed in the D1—D1′ and V3helices, which can serve as precise molecular identification markers. This study confirms the existence of a conflict between the current morphological species definition and molecular evidence, revealing that N. flagelliforme is not a natural monophyletic group and that cryptic species are highly probable. The molecular diversity pattern of N. flagelliforme in Northwest China revealed in this study not only clarifies its phylogenetic position, but also provides a standardized molecular identification ruler, offering scientific support for wild population resource surveys and germplasm resource bank construction.