ABSTRACT: Microbial mats are occasionally reported in thermal springs and information on such mats is very scarce. In this study, microbial mats were collected from two hot springs (Brandvlei (BV) and Calitzdorp (CA)), South Africa and subjected to scanning electron microscopy (SEM) and targeted 16S rRNA gene amplicon analysis using Next Generation Sequencing (NGS). Spring water temperature was 55°C for Brandvlei and 58°C for Calitzdorp while the pH of both springs was slightly acidic, with an almost identical pH range (6.2-6.3). NGS analysis resulted in a total of 4943 reads, 517 and 736 OTUs for BV and CA at, respectively, a combined total of 14 different phyla in both samples, 88 genera in CA compared to 45 in BV and 37.64% unclassified sequences in CA compared to 27.32% recorded in BV. Dominant bacterial genera in CA microbial mat were Proteobacteria (29.19%), Bacteroidetes (9.41%), Firmicutes (9.01%), Cyanobacteria (6.89%), Actinobacteria (2.65%), Deinococcus-Thermus (2.57%), and Planctomycetes (1.94%) while the BV microbial mat was dominated by Bacteroidetes (47.3%), Deinococcus-Thermus (12.35%), Proteobacteria (7.98%), and Planctomycetes (2.97%). Scanning electron microscopy results showed the presence of microbial filaments possibly resembling cyanobacteria, coccids, rod-shaped bacteria and diatoms in both microbial mats. Dominant genera that were detected in this study have been linked to different biotechnological applications including hydrocarbon degradation, glycerol fermentation, anoxic-fermentation, dehalogenation, and biomining processes. Overall, the results of this study exhibited thermophilic bacterial community structures with high diversity in microbial mats, which have a potential for biotechnological exploitation.