ABSTRACT Strains of Lactobacillus salivarius are increasingly employed as probiotic agents for humans or animals. Despite the diversity of environmental sources from which they have been isolated, the genomic diversity of L. salivarius has been poorly characterized, and the implications of this diversity for strain selection have not been examined. To tackle this, we applied comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) to 33 strains derived from humans, animals, or food. The CGH, based on total genome content, including small plasmids, identified 18 major regions of genomic variation, or hot spots for variation. Three major divisions were thus identified, with only a subset of the human isolates constituting an ecologically discernible group. Omission of the small plasmids from the CGH or analysis by MLST provided broadly concordant fine divisions and separated human-derived and animal-derived strains more clearly. The two gene clusters for exopolysaccharide (EPS) biosynthesis corresponded to regions of significant genomic diversity. The CGH-based groupings of these regions did not correlate with levels of production of bound or released EPS. Furthermore, EPS production was significantly modulated by available carbohydrate. In addition to proving difficult to predict from the gene content, EPS production levels correlated inversely with production of biofilms, a trait considered desirable in probiotic commensals. L. salivarius displays a high level of genomic diversity, and while selection of L. salivarius strains for probiotic use can be informed by CGH or MLST, it also requires pragmatic experimental validation of desired phenotypic traits.