In this study, fatty-acid content and patterns were analyzed in order to distinguish Nostoc flagelliforme, an edible terrestrial cyanobacterium, from other Nostoc species and representatives typical of its close neighbors (genera Anabaena, Microcystis, and Synechococcus). According to the Kenyon-Murata classification system, all the Nostoc species were assigned to Group II due to the presence of C18:20 and C18:30, and the absence of C18:3n6. Hierarchical cluster analysis was also employed to separate N. flagelliforme and other Nostoc species or strains. A dendrogram calculation of all fatty-acid components manifested phenetic characteristics, showing that the degree of relatedness of two strains of N. flagelliforme aggregated them within a small subgroup. Another dendrogram, calculated from seven comprehensive parameters (including ratios of different fatty-acid categories, degree of fatty-acid unsaturation, etc.), also clearly delimited the minute difference in fatty-acid profiles between the tested organisms. Our results suggest that profiling fatty acids could be a useful approach in the taxonomic or phylogenetic study of the genus Nostoc and might serve as a valuable supplement to the current morphology-based classification system.