Abstract Taxonomic profiling is a crucial task in microbiome research that aims at detecting and quantifying the relative abundance of microbes in biological samples. However, in many environments, varying fractions of microbial species still lack a sequenced genome and remain unaccounted for during taxonomic profiling based on shotgun metagenomic data. We present mOTUs3, a software tool which uses phylogenetic marker genes to profile metagenomes for 33.5k species-level operational taxonomic units, two thirds of which are currently not represented by sequenced genomes.