Antarctica is one of the most pristine environment in the world with a little human influence, where the anthropic activities are limited to military and scientific purposes. James Ross archipelago is an island group located in the NE of Antarctic Peninsula and high number of freshwater systems could be found in the archipelago’s free-ice areas. Despite of the rough environmental conditions, these systems allow a complex microbial mats development. The community composition of these ecosystems is diverse and it is result of predominant species and the environment surrounding the mats. Given the biological importance of these communities detailed studies of the mats diversity are needed. For that reason, in this work we have proposed to study using high-throughput sequencing technologies (Ilumina amplicon sequencing), the microbial mats diversity in three lakes differing in geochemical parameters of James Ross and Vega Island. The experimental approach included 16S rRNA V3-V4 region sequencing and QIIME2 analysis of reads.