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This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus.A primer set (CbsA2F–CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI–Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1–A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F–CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI–Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq.A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly.To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus.