The large sulfur-oxidizing bacteria (LSB) are a morphologically diverse group of sulfide-oxidizing Beggiatoaceae (Gammaproteobacteria), including free-living and sheathed filaments, vacuolated and nonvacuolated species, and some of the largest known bacteria. The family Beggiatoaceae includes the genera Beggiatoa, Thioploca, Thiomargarita, Cand. Marithrix, and several other Candidatus groups. They are found at sulfidic/oxic interfaces in both marine and freshwater settings, including hydrothermal sites, and may store nitrate, elemental sulfur, polyphosphate, and/or carbohydrates, allowing them to tolerate environmental fluctuations or to migrate between sulfidic and oxidizing conditions. Only two species are in cultivation, both nonvacuolated Beggiatoa spp. from freshwater environments, but cells from several vacuolated species have been directly collected for genome sequencing. Comparison of these genomes with those of related Gammaproteobacteria reveals several changes that may have been key to the evolution of the LSB, in particular the loss of canonical genes for septum formation and for DNA replication initiation by DnaA. The evolutionary origin and mechanism of division of the central vacuoles found in some LSB remains a puzzle; we suggest that the acquisition of dynamin-family proteins may have been one key step. LSB genetic traits related to DNA replication have parallels to those in the Cyanobacteria, another morphologically diverse group with which the LSB appear to have a history of gene exchange. We conclude with a proposed model for the evolution of the LSB and suggest observations and experiments that could be used to test it.