Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 15 6 2023 11 20 723 733 Atif Wani Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Jalandhar, Punjab, India Suhail Ahmad Department of Remote Sensing and GIS, University of Jammu, Jammu, India Juliana Heloisa Pinê Américo-Pinheiro Department of Forest Science, Soils and Environment, School of Agronomic Sciences, São Paulo State University (UNESP), Botucatu, Brazil; Department of Environmental Sciences, Graduate Program in Environmental Sciences, Brazil University, São Paulo, Brazil Nazli Tizro Department of the Environment, College of Natural Resources and Environment, Science and Research Branch, Islamic Azad University, Tehran, Iran Reena Singh Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Jalandhar, Punjab, India 2023 08 11 2023 10 10 Background and Objectives: Rinaie Marwah hot spring Kishtwar (RMHSK) is one of the geothermal springs located at 33°51′51′′N 75°32′07′′E with an elevation of 2134 meters above sea level in Jammu and Kashmir, India. We aimed to study the microbial diversity of this geothermal spring using metagenomics. Materials and Methods: In the present study, physiochemical parameters including temperature (65-75oC), pH (6. 9-8. 8), hardness (250 ppm), and mineral content was measured along with the microbial diversity using Illumina MiSeq metagenome-based 16s amplicon sequencing (V3-V4). The sequence reads were classified taxonomically into 31 phyla, 71 classes, 152 orders, 256 families, 410 genus, and 665 species. QIIME 2 (Quantitative Insights into Microbial Ecology), an extensible, powerful, and decentralized analytical tool, was used for taxonomic analysis. Results: Bacteroidota (32. 57%) was the dominant phylum, Bacteroidia (32. 51%) the dominant class, Bacteroidales (16. 6%) the dominant order, and Lentimicrobiaceae (14. 23%) was the dominant family per the abundance analysis. Shannon (2. 28) and Chao 1 (87. 0) diversity indices support the existence of higher microbial diversity in RMHSK (50717 OTUs). Conclusion: The microbial diversity of RMHSK is reported for the first time through a metagenomic study. Identification of microorganisms with characteristics that are relevant to industries. https://ijm.tums.ac.ir/index.php/ijm/article/view/4354 https://ijm.tums.ac.ir/index.php/ijm/article/download/4354/1618