Abstract. Ardhi A, Sidauruk AN, Suraya N, Pratiwi NW, Pato U, Saryono. 2020. Molecular identification of amylase-producing thermophilic bacteria isolated from Bukit Gadang Hot Spring, West Sumatra, Indonesia. Biodiversitas 21: 994-1000. Amylase is one of the hydrolytic enzymes that is widely used in a wide number of industrial processes such as food, fermentation, textile, paper, detergent, and pharmaceutical industries. Amylase produced by thermophilic bacteria may be thermostable, which is very beneficial in several applications requiring high temperature, for example, the process of gelatinization, liquefaction, and saccharification are performed in high temperature involved in the starch processing. In this study, the amylase-producing ability of thermophilic bacteria isolated from Bukit Gadang hot spring, West Sumatra, Indonesia, was checked and followed by molecular identification. Thirteen isolates that were successfully isolated from the hot springs were microscopically and macroscopically characterized, biochemically tested, and determined their amylase enzyme activity both qualitatively and quantitatively. The isolate that performed the best amylase activity was identified using the molecular technique. The DNA sequencing was carried out in 16S rRNA and continued with BLAST search for species identification. The result of molecular identification showed that the isolate with the best amylase activity was identified as Bacillus licheniformis. The optimum amylase production (231.33 U/ml) and the best enzyme-specific activity (101.79 U/mg) were obtained at the incubation time of 36 hours.