<p>Despite being ubiquitous in oceans and important in marine biogeochemical cycles, planktonic archaea in the Southern Ocean (SO) remain poorly characterized. Although high-throughput sequencing (HTS) approaches based on 16S ribosomal RNA (rRNA) genes have been used widely to study the diversity and composition of microbial community in natural environments, primer-set selection is critical because of amplicon-sequencing bias during metabarcoding. Here, using surface-seawater samples collected from the area between the South Shetland and South Orkney Islands, Antarctica, we compared primer sets Arch349F/Arch806R, 515F-Y/926R, and 524F/Arch958R, which target different 16S rRNA gene hypervariable regions to identify the best one for studying planktonic archaeal communities. With much lower number of bacteria-related sequences, primer set 524F/Arch958R showed higher values of archaeal operational taxonomic units (OTUs) as well as alpha-diversity indices, indicating that this primer set was more specific for detecting archaeal species and could be helpful to obtain more comprehensive information on the archaeal community compositions compared to other two primer sets. Compared with primer set Arch349F/Arch806R revealing four phyla (Halobacteriota, Methanobacteriota, Thermoplasmatota, and Thermoproteota) detected in seawater, additional archaeal phyla were observed by 515F-Y/926R (Asgardarchaeota and Nanoarchaeota) and 524F/Arch958R (Micrarchaeota). In spite of the differences in archaeal community compositions observed among the three investigated primer sets, ammonia-oxidizing (e.g., Nitrososphaeria) and methane-producing (e.g., Methanobacteria, Methanomicrobia, and Methanosarcinia) archaea were the main groups detected in the surface seawater, indicating the ecological role of planktonic archaea in carbon and nitrogen cycling in the upper waters of the SO. These results underscore the importance of primer-set selection when studying archaeal community diversity and composition in the Antarctic SO.</p>