The present study was conducted to isolate, identify, characterize and to determine the enzymatic activities of the thermophilic Geobacillus species from five Jordanian hot springs. Based on phenotypic characters, eight thermophilic isolates were identified and belonged to the genus Geobacillus. The Geobacillus isolates were abundant in all investigated hot springs. The optimal temperature for growth of the isolates was 60 to 65°C and the optimal pH was 6 to 8. Colonies were light yellow circular to rhizoid. The bacterial cells were Gram positive rods and endospore forming. All isolates produced amylase, caseinase, alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), α-Galactosidase, β-Glucuronidase, β-Glucosidase, and N-Acetyl-β-glucosaminidase. Seven isolates produced leucine and valine arylamidases and five isolates produced naphthol-AS-B1- phsphohydrolase. Lipase (C14) activity from two isolates and α-chymotrypsin activity from three isolates were also detected. The phenotypic characterization of those isolates was confirmed by genotypic method using 16S rDNA sequence analysis. Maximal homology of all eight isolates to genus Geobacillus was observed. Five of these isolates showed greater than 98% homology with Geobacillus stearothermophilus and one isolate showed 100% homology with Geobacillus thermoglucosidasius. Therefore, 16S rRNA gene sequence analysis can be considered as a valuable genotypic tool for the identification and characterization of thermophilic bacteria at genus level. Moreover, enzymatic products of those isolates could receive considerable attention due to their potential applications in biotechnology.Keywords: Thermophiles, Geobacillus, hydrolytic enzymes, hot spring, 16S rRNA
