Aerobic spore -forming bacteria of the order Bacillales represent a highly diverse and ubiquitous group that includes organisms that cause foodborne illnesses and food spoilage. Classical microbiological and biochemical identification of members of the order Bacillales represents a challenge due to the diversity of organisms in this group as well as the fact that the taxonomic assignment of many validly named species in this group is not consistent with their genomic characteristics. DNA-sequencing-based tools, on the other hand, have been shown to enable fast, cost-effective, and more reliable identification and characterization of Bacillales isolates. In comparison to 16S rDNA, rpoB was shown to better discriminate between Bacillales isolates and to allow for taxonomic assignment to the species and subspecies levels. However, the lack of a publicly accessible rpoB database, as well as the lack of standardized protocols for rpoB-based typing and strain identification, is a major challenge. Here, we report (i) a curated DNA sequence database and online tool for rpoB-based subtype classification and identification of Bacillales isolates; and (ii) standardized protocols for PCR-amplification and sequencing of rpoB from Bacillales isolates. The curation of a rpoB database will facilitate widespread application of the rpoB sequence-based approach for the characterization of aerobic and facultative anaerobic spore-forming Bacillales isolates. Specifically, we curated a database of DNA sequences for a 632-nt internal variable region within the rpoB gene from the Bacillales representative reference type strains and a large number of isolates that we have previously isolated and characterized in the frame of multiple pathogen environmental monitoring projects. As of May 2021, the rpoB database contains more than 8,350 rpoB sequences representing 1,902 distinct rpoB allelic types that can be classified into 160 different genera; this database also includes 1,129 rpoB sequences for representative reference type strains of the order Bacillales as available on May 2021 in the NCBI database. The rpoB database is integrated into the online Food Microbe Tracker platform (www.foodmicrobetracker.com) and can be queried using the integrated BLAST tool to initially subtype and taxonomically identify aerobic and facultative anaerobic spore-formers. While whole-genome sequencing is increasingly used for the characterization of bacterial isolates, this rapid and standardized single gene sequence-based scheme provides a valuable tool as it allows for rapid and cost-effective initial isolate characterization, which often is essential to promptly detect and identify foodborne pathogens and foodspoilage bacteria. In addition, the database and primers described here can also be adopted for target metagenomics approaches that include rpoB as a target, improving discriminatory power and identification over what can be achieved using 16S rDNA as a target.