Four strains of a Gram-stain-positive, coccoid, catalase-positive, non-motile bacterium were recovered from nasal lavage samples collected from children in Wisconsin during the Spring of 2008. These strains, designated RSM42T, RSM292, RSM386 and RSM407, were subjected to a comprehensive biochemical and polyphasic taxonomic investigation. Despite the novel bacterium sharing 99.6% 16S rRNA gene sequence identity with Rothia mucilaginosa 5762/67T, BLAST+ average nucleotide identity, MUMmer3 average nucleotide identity and digital DNA–DNA hybridization values of 91.3%, 91.9% and 43.1%, respectively, were below the cut-off values routinely used for species demarcation. Consistent with these findings, phylogenetic and pangenomic comparisons indicated that RSM42T, RSM292, RSM386 and RSM407 form a separate lineage within the genus Rothia. Strain RSM42T is further distinguished from R. mucilaginosa 5762/67T by its unique ability among Rothia species to use fructose-6-phosphate as a sole carbon source. RSM42T also exhibits an enzyme activity profile consistent with R. mucilaginosa, as it is positive for valine arylamidase and negative for C4 esterase, β-glucosidase, pyrazinamidase and trypsin, a combination not observed in other Rothia species. The major fatty acids were anteiso-C15:0 (44.2%) and iso-C16:0 (14.4%), and the moderate fatty acids were anteiso-C13:0 (2.3%), iso-C14:0 (6.0%), C14:0 (2.3%), iso-C15:0 (5.9%), C15:0 (1.9%), C16:0 (9.3%) and anteiso-C17:0 (9.5%). The major polar lipids were aminoglycolipid and diphosphatidylglycerol. Based on biochemical, phylogenetic, genotypic and chemotaxonomic criteria, these isolates represent a novel species within the genus Rothia, closely related to R. mucilaginosa, for which the name Rothia similimucilaginosa sp. nov. is proposed. The type strain is RSM42T (=ATCC TSD-447T=DSM 118581T).