The present study was conducted to identify and characterize the thermophilic bacteria isolated from five hot springs, namely, Sinkosh, Singha, Bhurung, Ratopani, and Paudwar located in Myagdi district, Nepal, using phenotypic and genotypic methods. The hot spring has temperature 42-62°C and pH 6.5-6.8. Isolation of thermophiles was done using simple enriched nutrient broth media at 60°C. Selected strains were screened for thermostable enzymes; cellulase, hemicellulase, amylase, protease, gelatinase, and lipase using substrates carboxymethylcellulose, xylan, soluble starch, casein, gelatin, and Tween (20, 40, 60, or 80), respectively. The bacteria were grouped into 16 groups based on morphological and biochemical characteristics. 16S rRNA sequence analysis of 16 isolates and phylogenetic analysis showed a cluster of five distinct taxonomic groups. The groups were identified as genus Anoxybacillus, Aeribacillus, Brevibacillus, Bacillus, and Geobacillus, based on ≥95% similarity with reference strains. This is the first study that reports Anoxybacillus sp., Brevibacillus sp., and Aeribacillus sp. from the hot springs of Nepal.