A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria. Display omitted •Construction of two ordered gene deletion mutant libraries of B. subtilis•Defined essential and auxotrophic gene sets in B. subtilis•High-throughput methods for transformation and double-mutant analysis•Genome-wide screening of growth, competence, and sporulation Koo et al. have constructed two complete ordered single-gene deletion mutant libraries of the model Gram-positive model bacterium, B. subtilis, and developed new high-throughput methodologies to enable facile screening. They have assessed gene essentiality, auxotrophy, competence, and sporulation genome-wide. These libraries and methods provide versatile resources for the study of gene functions, pathway connections, and their regulation.