One of the most significant challenges for detecting microbial life in thermal springs by conventional techniques such as culturing is these places’ physicochemical (temperature, heavy metal content, pH, etc.) conditions. Data from several studies suggest that high-throughput DNA sequencing technologies can be used to perform more accurate and detailed microbiome analyses. The primary aim of this paper was to determine the microbiome in the thermal source by metabarcoding environmental DNA isolated from four different sources and reveal the reflection of differences caused by temperature and chemical content on the microbiome. DNA was extracted from water filtered with enclosed filters and using the Illumina high-throughput sequencing platform, V3 and V4 regions of the 16S rRNA gene were sequenced. The results showed a correlation between physicochemical conditions and microorganism composition of four different thermal springs. Springs with extremely high temperature (89–90 °C) were dominated by hyperthermophiles such as Hydrogenobacter and Thermus, while a spring with a high temperature (52 °C) was dominated by thermophiles such as Thermoanaerobaculum and Desulfurispora, and a spring with a low temperature (26 °C) and high salinity was dominated by halophiles and sulfur-oxidizers such as Hydrogenovibrio and Sulfirimonas. With this research, we observed many manipulable steps according to the work of interest. This study sought to obtain data that will help decide the right gene region and choose the optimal bioinformatic pipeline.