Four Gram-positive cocci were isolated from the cerebrospinal fluid or blood of four different patients, but they could not be identified by an automated conventional identification system, so they were identified using cellular fatty acid (CFA) composition analysis and 16S rRNA gene sequencing analysis. Of these, two strains (SMC-A2662 and SMC-A5889), which were previously supposed to be Rothia dentocariosa according to the API Coryne system, were identified as Rothia aeria by the 16S rRNA gene analysis. SMC-A608, which was unidentified by both the VITEK2 and API Coryne systems, was identified as Rothia mucilaginosa. The one remaining SMC-2244T was distinguished from the other Rothia species by its biochemical profile, its CFA composition and its 16S rRNA gene sequence. Phylogenetic analysis showed that it was closely related to Rothia nasimurium but the 16S rRNA gene sequence dissimilarity of 1.8% was enough to differentiate it from R. nasimurium. Based on both the phenotypic and phylogenetic evidence, we propose a new species name for this bacterium, Rothia arfidiae sp. nov. The results of this study show that several Rothia species were isolated from human and we have identified them using 16S rRNA gene sequences.