Lactobacillus brevis is the major spoiler in beer, affecting product quality and causing economic losses in breweries. Although several genes involved in beer-spoilage have been identified, there are still unresolved questions about which genes that are associated with growth in beer and the potential roles of these genes. In this study, 21 Lactobacillus brevis strains were tested for beer spoilage potential; three beer-tolerant and three beer-sensitive strains were selected for comparative genomic analysis. One of the tolerant strains was exposed to growth conditions containing novobiocin that favor loss of plasmids, and subsequently became more sensitive to beer as determined by growth experiments. The genetic difference between the wildtype and the sensitive mutant confirmed that the previously identified beer (hop)-tolerance genes horA and hitA, which are usually localized on plasmids, play important roles in beer spoilage. Interestingly, horA and hitA were present in another sensitive strain whereas these genes were absent in another tolerant strain. This indicates that a beer-spoilage phenotype cannot be easily identified from the presence of a few reported beer-spoilage genes. The potential roles of additional genes involved in beer tolerance were discussed, including a ClpX protease and a manganese transporter different from hitA.