Stanieria strains isolated from hot springs in Indonesia were identified on the basis of the strains’ morphological, chemotaxonomy, and molecular characteristics. Based on the cell shape, the sizes of vegetative cells and the existence of baeocytes (endospores) observed in these strains, are the factors commonly considered in characterizing Stanieria. However, Stanieria strains are difficult to identify by morphological analysis alone. The results of the MALDI-TOF analysis of whole cell protein showed that the mass spectral peaks of the six Stanieria strains were all found at 56 kDa. The dendogram plot based on mass spectral peaks shows that all strains belong to one cluster, indicated that they belong to the same genus. The molecular properties considered were molecular sequences of the 16S rRNA gene and the internal transcribed spacer sequences between the 16S and 23S rRNA genes. Identification at the genus level based on molecular data is in agreement with MALDI-TOF data. The 16S rRNA gene sequence similarity indicated that the six strains are closest to the species S. cyanosphaera, varying between 95% and 99%. The phylogenetic tree of six Stanieria strains and closely related species obtained from DNA database Genbank was constructed based on 16S rRNA gene partial sequence data. Although all six strains Stanieria from hot springs are closely related to Stanieria cyanosphaera, the phylogenetic tree showed that Stanieria strains from hot springs are grouped into two clusters outside the S. cyanosphaera reference strain cluster. They may also be composed of two different species, distinct from S. cyanosphaera.