Abstract Objectives: To developed a 16S rRNA PCR-denaturing gradient gel electrophoresis to identify casei group of Lactobacillus at species level Methods: PCR-denaturing gradient gel electrophoresis (PCR-DGGE) has been developed based on the 16S rRNA. Two pairs of specific primers at V2-V3 region, HDA1-GC, F165-GC and HDA2 have been used. The bacterial strains included in this study were L. casei ATCC 393, L. casei CCUG 31610, L. paracasei CCUG 32212, L. rhamnosus ATCC 7469, L. zeae CCUG 35515. In addition, 60 clinical strains of L. casei/paracasei/rhamnosus which previously identify using 16S rRNA PCR-RFLP were included. Results: The migration pattern of the PCR amplicons of L. paracasei CCUG 32212 and clinical strains of L. paracasei indicated that there were more than one copies of 16S rRNA gene from primer HDA1-GC and HDA2, and this has been confirmed by DNA sequencing. L. casei could be differentiated from L. rhamnosus by the migration pattern of the PCR amplicons of F165-GC and HDA2. Conclusions: DGGE provides a promising tool for identification the casei group of Lactobacillus at species level.