Abstract:The majority of microbial species in the environment remains uncultivated, called uncultivated microorganisms or microbial "dark matter". Unraveling the mysteries of these microbial "dark matter" is especially important to understand the diversity of microbes and their metabolic characteristics. These data can provide insights into the microbes involved in ecological processes, and insights into the early diversification of microbial lineages and the evolution of Bacteria and Archaea. DNA genome sequences of microbial "dark matter" could be recovered from the environment samples by population binning of metagenomics and single-cell genomics, independently or combined synergistically. The metabolic potential could be predicted based on bioinformatics analysis. In this mini-review, we briefly introduce the methods and challenges in this area, summarize the main groups of microbial "dark matter" that has been explored, and indicate the future research opportunities.