Four isolates, designated, RG-1T, RG-3T, RG-6 and RG-8 with optimum growth temperatures around 60 °C were recovered from a hot stream near Ribeira Grande on the Island of São Miguel in the Azores. These strains were strictly aerobic, catalase and oxidase positive. The organisms could be divided into two groups; one comprising strains RG-1T and RG-6, the other formed by strains RG-3T and RG-8, with distinctive phenotypic and chemotaxonomic characteristics. Major fatty acids varied between the groups, but all contained iso-branched components such as iso-C16:0 10-methyl and iso-C17:0 10-methyl. Analysis of the 16S rRNA gene shows the strains to cluster with species of the genus Rubrobacter, specifically with Rubrobacter xylanophilus to which they have pairwise sequence similarity in the range of 98.4–98.6%. The DNA–DNA hybridization result show that strains RG-1T and RG-3T share 72% reassociation. Strains RG-1T and RG-3T displayed extreme tolerance to desiccation when compared to other species of the genus. Based on genotypic, physiological and biochemical characteristics we describe two new species: Rubrobacter calidifluminis sp. nov., represented by strain RG-1T (=CECT 8308T =JCM 19154T) and Rubrobacter naiadicus sp. nov. represented by strain RG-3T (=CECT 8310T = JCM 19155T). The description of the species Rubrobacter bracarensis [16] is emended to correct the fatty acid composition of these organisms. The discovery of new fatty acids in these species requires emendation of the description of the genus Rubrobacter.