A polyphasic study revealed taxonomic heterogeneity among reference strains of the species Lactobacillus brevis. Representative strains of L. brevis and related taxa were genomically investigated by partial sequence analysis of the housekeeping gene encoding the alpha subunit of the phenylalanyl-tRNA synthase (pheS). Species-specific clusters were delineated for all taxa studied except for two L. brevis strains, LMG 11494 and LMG 11984T, which occupied a distinct position. Their phylogenetic allocation was determined using 16S rRNA gene sequence analysis and showed that both strains belong to the Lactobacillus buchneri group with nearest neighbours Lactobacillus hammesii and L. brevis (sequence similarities of 99.2 and 98.1%, respectively). The strains LMG 11494 and LMG 11984T, isolated from cheese and wheat, respectively, yielded 99.9% sequence similarity among each other. Further genotypic and phenotypic study, including DNA-DNA hybridizations, clearly demonstrated that both strains represent a single novel taxon for which the name Lactobacillus parabrevis sp. nov. is proposed with the type strain LMG 11984T (= ATCC 53295T).