AbstractAntarctic lakes have generally simple periphyton communities when compared with those of lower latitudes. To date, assessment of microbial diversity in Antarctica has relied heavily on traditional direct observation and cultivation methods. In this study, sterilized cotton baits were left submerged for two years in two lakes on King George Island and Deception Island, South Shetland Islands (Maritime Antarctic), followed by assessment of diversity by metabarcoding using high-throughput sequencing. DNA sequences of 44 taxa belonging to four kingdoms and seven phyla were found. Thirty-six taxa were detected in Hennequin Lake on King George Island and 20 taxa were detected in Soto Lake on Deception Island. However, no significant difference in species composition was detected between the two assemblages (Shannon index). Our data suggest that metabarcoding provides a suitable method for the assessment of periphyton biodiversity in oligotrophic Antarctic lakes.