Genomics promises comprehensive surveying of genomes and metagenomes, but rapidly changing technologies and expanding data volumes make evaluation of completeness a challenging task. Technical sequencing quality metrics can be complemented by quantifying completeness of genomic datasets in terms of the expected gene content of Benchmarking Universal Single-Copy Orthologs (BUSCO, http://busco.ezlab.org). The latest software release implements a complete refactoring of the code to make it more flexible and extendable to facilitate high-throughput assessments. The original six lineage assessment datasets have been updated with improved species sampling, 34 new subsets have been built for vertebrates, arthropods, fungi, and prokaryotes that greatly enhance resolution, and datasets are now also available for nematodes, protists, and plants. Here we present BUSCO v3 with example analyses that highlight the wide-ranging utility of BUSCO assessments, which extend beyond quality control of genomics datasets to applications in comparative genomics analyses, gene predictor training, metagenomics, and phylogenomics.