A major fraction of biodiversity on Earth resides in soils. This diversity consists of prokaryotes (bacteria and archaea) and eukaryotes (fungi, protists and metazoa). Especially the prokaryotic diversity is almost entirely studies using molecular tools, with a recent transfer also to eukaryotes.. Eukaryotes differ from prokaryotes in many additional characteristics such as genomic organization and mode of evolution, which together determine the methods to study their diversity. As such, approaches that have been designed for prokaryotes like environmental sequencing cannot simply be transferred to eukaryotes. Indeed, methodological and analytical differences between domains range from how the basic diversity units are measured to interpretation of community taxonomic composition and quantitative data. Jurburg et al. (“All together now: Limitations and recommendations for the simultaneous analysis of all eukaryotic soil sequences” Molecular Ecology Methods, 2021 https://doi.org/10.1111/1755-0998.13401) have highlighted some important eukaryote-specific technical issues that need consideration into account in DNA-based studies. We here highlight additional domain-specific considerations that should be taken into account in eukaryote-focused studies. Only that will allow to reliably and comprehensively unravel the entire eukaryotic biodiversity in soils.