The present study was conducted to determine the phenotypic and genotypic characterization of thermophilic bacteria isolated from Pasinler hot spring, Erzurum, Turkey. Fatty acid profiles, BOX PCR fingerprints, and 16S rDNA sequence data were used for the phenotypic and genotypic characterization of thermophilic bacteria. Totally 9 different bacterial strains were selected based on morphological, physiological, and biochemical tests. These strains were characterized by molecular tests including fatty acid and BOX profiles, and 16S rDNA sequence. The data of fatty acid analysis showed the presence of 14 different fatty acids in the 9 bacterial strains examined. Additionally, 5 of these fatty acids, 15:0 iso, 15:0 anteiso, 16:0 iso, 17:0 iso, and 17:0 anteiso fatty acids, were found in all isolates. Based on fatty acid profiles, it was determined that the bacterial strains were classified into 2 phenotypic groups, and these data were confirmed by BOX PCR genomic fingerprint profiles and 16S rDNA sequence analysis. The first group, identified as Bacillus licheniformis, was represented by 4 strains, and the second group, identified as Aeribacillus pallidus, was represented by 5 strains.