Permafrost soils, which contain one of Earth’s largest terrestrial carbon stocks, are vulnerable to thaw and microbial decomposition, exacerbating climate change. Advancements in sequencing technologies have facilitated the identification and functional profiling of microbial communities in permafrost, but DNA extraction from these soils is challenging due to their high microbial diversity and low biomass. This study assessed the effectiveness of the DNeasy PowerSoil Pro kit in extracting DNA from permafrost samples and found that it produced significantly different results than the discontinued DNeasy PowerSoil kit. The study highlights the importance of consistent DNA extraction methods in permafrost studies.