Lactobacilli belong to the genus Lactobacillus, the largest genus among the lactic acid bacteria (LAB). They are abundant in plant material and food resources, or they may inhabit niches in or on the bodies of humans and animals, as commensals. Lactobacilli of food origin are commercially important in the production of dairy products, fermented meats, vegetables, and sourdough, and many of their properties have been well studied. Commensal lactobacilli are good candidates for development as probiotics. In recent years, the general biology and host interaction mechanisms of commensal lactobacilli have attracted great interest. Although the metabolic pathways, predicted gene functions, and some phenotypic traits, of commensal lactobacilli can be inferred or deduced to an extent by the growing number of Lactobacillus genome sequencing project, various genetic tools are still required to confirm their phenotypic properties and biological traits. The current state of the art with respect to the available complement of genetic tools including genomic resources, and more traditional approaches to investigate the biology of commensal lactobacilli, will now be reviewed.