Two strains, KBL13(T) and GBL13, were isolated as one of intestinal lactobacilli from the faecal specimens from different thoroughbreds of the same farm where they were born in Hokkaido, Japan. They were Gram-positive, facultatively anaerobic, catalase-negative, non-spore-forming and non-motile rods. KBL13(T) and GBL13 homofermentatively metabolize glucose, and produce lactate as the sole final product from glucose. The 16S rRNA gene sequence, DNA-DNA hybridization, DNA G + C content and biochemical characterization indicated that these two strains, KBL13(T) and GBL13, belong to the same species. In the representative strain, KBL13(T) the DNA G + C content was 34.3 mol%. Lactobacillus salivarius JCM 1231(T) (=ATCC 11741(T); AF089108) is the type strain most closely related to the strain KBL13(T) as shown in the phylogenetic tree, and the 16S rRNA gene sequence identity showed 96.0% (1425/1484 bp). Comparative 16S rRNA gene sequence analysis of this strain indicated that the two isolated strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives, L. salivarius, Lactobacillus aviarius, Lactobacillus saerimneri and Lactobacillus acidipiscis. DNA-DNA reassociation experiments with L. salivarius and L. aviarius confirmed that KBL13(T) represents a novel species, for which the name Lactobacillus hayakitensis sp. nov. is proposed. The type strain is KBL13T (=jCM 14209(T) =DSM 18933(T)).