show Abstracthide AbstractStratified lakes and ponds that feature steep oxygen gradients are known to be significant net sources of greenhouse gases and hotspots in the carbon cycle. Despite their significant biogeochemical roles, the resident microbial communities, especially in the oxygen depleted hypolimnetic compartments are poorly known. In this paper, we present a comprehensive metagenome dataset including shotgun metagenomes from around 30 stratified lakes and ponds mainly located in the boreal and subarctic regions, but also including one reservoir from tropical Puerto Rico and one Swiss lake from the temperate zone. For most lakes and ponds, the data includes a vertical sample set spanning the oxic surface waters to anoxic bottom layers. In addition to the metagenomic sequences, the dataset includes environmental variables for the samples, such as oxygen, nutrient and carbon concentrations. The dataset is ideal for further exploring the microbial taxonomic and functional diversity in freshwater environments and climate change impacts on the functioning of these ecosystems.