Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form-genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form-genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form-genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form-genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form-genus Cyanothece were observed to be in a close relationship with our isolates.