Abstract: Objective To establish a PCR assay for serotyping of Shigella flexneri serotype Xv. Methods Based on O-antigen structure of S. flexneri serotype Xv, three primer pairs targeting O-antigen synthesis gene wzx, antigenicity 7;8 determined gene gtrX and MASF IV-1 specific gene opt were designed and used in a PCR assay to establish a molecular assay for serotyping of S. flexneri serotype Xv. A total of 139 clinical isolates of S. flexneri were serotyped with this assay and the results were compared with slide agglutination method. Results A PCR assay targeting O-antigen modification genes of S. flexneri serotype Xv was established. The PCR assay contains three primer pairs, which can identify serotype Xv(all target genes are positive)in one reaction and can distinguish serotype Xv from other known 18 serotypes of S. flexneri. No ampliflcation products of non Shigella and diarrhea related isolates were detected with the assay established. A total of 38 serotype Xv strains were detected from 139 clinical isolates of S. flexneri. The result was consistent with that of slide agglutination method. Conclusion This PCR assay is rapid and specific and can be used in Shigella detection and surveillance.