A biodiversity study on lactic acid bacteria (LAB) occurring in traditional Belgian sourdoughs resulted in the isolation of two Lactobacillus isolates, LMG 23583^T and LMG 23584, that could not be assigned to any recognized LAB species. The two isolates were initially investigated by means of phenylalanyl-tRNA synthase (pheS) gene sequence analysis and were found to occupy a separate position relative to recognized Lactobacillus species present in the pheS database. Subsequently, their phylogenetic affiliation was determined by 16S rRNA gene sequence analysis, indicating that the two isolates belong to the Lactobacillus buchneri species group with Lactobacillus zymae, Lactobacillus acidifarinae and Lactobacillus spicheri as closest relatives. Whole-cell protein analysis (SDS-PAGE) and amplified fragment length polymorphism fingerprinting of whole genomes confirmed their separate taxonomic status. DNA–DNA hybridization experiments, DNA G+C content, growth characteristics and biochemical features demonstrated that the two isolates represent a novel Lactobacillus species, for which the name Lactobacillus namurensis sp. nov. is proposed. The type strain is LMG 23583^T (=CCUG 52843^T).