DNA–DNA hybridization (DDH), the gold standard for bacterial species delineation, is a laborious method and the alternative, average nucleotide identity (ANI), a genomic sequence-derived parameter, is not applicable to non-sequenced species. A universal cut-off value to delineate bacterial species does not exist, yet a DDH value <70 % and ANI <95±0.5 % have proved useful in selected examples. We herein compare published values for DDH and ANI with sequence similarity of rpoB gene sequences retrieved from GenBank for strains of 230 bacterial species representative of 45 genera. Intraspecific rpoB sequence similarity was 98.2–100 %. We observed that an rpoB gene sequence similarity ≤97.7 % significantly correlated with a DDH value <70 % and an ANI value <94.3 %. An rpoB gene sequence similarity <85.5 % correlated with membership of different genera. When applied to fastidious and as-yet-uncultivated organisms lacking experimental DDH values, these cut-off values suggested that ‘Candidatus Blochmannia pennsylvanicus’ and ‘Candidatus Blochmannia floridarius’ may belong to different genera, that the different endosymbiotic Buchnera aphidicola organisms may belong to different genera and that, while the tsetse fly enteric symbiont Sodalis glossinidius may belong to the Enterobacteriaceae, the endocellular obligate symbiont Wigglesworthia glossinidia from the same host may belong to the group of as-yet-uncultivated gammaproteobacteria. rpoB gene sequence similarity provides an efficient supplement to DDH and ANI measurements to delineate bacterial species and genera, including delineation of as-yet-uncultivated, non-sequenced organisms.