As member of the lactic acid bacteria (LAB), the genus Lactobacillus represents a diverse number of species that play significant roles in the biopreservation of foods and commensals common within the human gastrointestinal (GI) tract. Certain species of Lactobacillus, particularly those of human origin, have been used as probiotic bacteria due to their health-promoting effects. A recent explosion of genomic information on lactobacilli has expanded our knowledge of metabolic capabilities and key gene features that are predicted to play important roles in niche adaptation and function. This review provides an overview of probiotic-related genome features and functional genomic studies that have linked genes to traits. Interspecies heterogeneity and niche-specialized adaptation among lactobacilli, as revealed by comparative genome analysis, are also discussed.