Extremophilic enzymes can function with high efficiency under extreme conditions. Thermophilic bacteria constitute an important source in terms of these extremophilic enzymes. The isolation and identification of these bacteria and enzymes are very important for industrial applications. Many methods are used for the identification of microorganisms. The Fatty Acid Methyl Esters (FAME) analysis that enables the comparison of the methylated fatty esters of microorganisms in addition to 16S rRNA-based molecular identification also takes a significant place in the taxonomy. In this study, we examined the hydrolytic enzyme (protease, amylase, and lipase) production ability of our 65 thermophilic isolates, and the FAME analysis of some isolates was performed. As a result of the study, 42 thermophilic isolates exhibited a weak or strong protease, amylase and lipase activity. However, 23 isolates showed no activities for these enzymes when cultured on skim milk, starch, and tributyrin agar. The fatty acid analysis data showed the presence of 35 different fatty acids in 18 bacteria strains examined. 16:0 fatty acid was found in all strains. As a result of the analyses carried out based on the fatty acid profiles, it was determined that the bacteria strains were divided into 12 phenotypic groups, but these data did not match the genotypic analysis results exactly.