Two Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strains, NCYUAST and BCRC 18859 (=NRIC 1947), were isolated from cow manure of Taiwan and coconut juice of Philippines, respectively. Comparative sequence analysis of 16S rRNA gene revealed that the novel strains were members of the genus Lactobacillus. These two strains had 100% of 16S rRNA gene sequence similarity and 98.6% of average nucleotide identity (ANI) value based on whole genome sequences. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (99.6% similarity), Lactobacillus paracasei subsp. paracasei (99.1%), L. paracasei subsp. tolerans (99.1%), Lactobacillus rhmnosus (99.0%) and 'Lactobacillus zeae' (99.7%) were the closest neighbors to these novel strains. The results of phenotypic and chemotaxonomic characterization, multilocus sequence analysis (MLSA) based on the sequences of three housekeeping genes (dnaK, pheS and yycH), whole-genome sequence (WGS)-based comparison by ANI and in silico DNA-DNA hybridization (isDDH), species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses demonstrated that the novel two strains represented a single, novel species within the L. casei group, for which the name Lactobacillus chiayiensis sp. nov., is proposed. The type strain is NCYUAST (=BCRC 81062T=NBRC 112906T).