[Objective] Based on the λ Red recombination system,a two-step PCR method was developed to delete small non-coding RNA (sRNA) and large chromosomal fragment in YYersinia pestis.[Methods] Two PCR procedures were done to amplify product formed of a kanamycin resistance gene flanked by long (600-1000 bp) homology extensions.The PCR fragment carrying a kanamycin resistance gene flanked by regions homologous to the target locus was electroporated into a recipient 201 strain of Yersinia pestis expressing the homologous recombination system encoded by plasmid pKD46,which promoted the replacement of the target gene with kanamycin resistant fragment.Finally,the recombinant clones were identified by PCR.[Results] The homologous extensions of 600-1000 bp were constructed by two PCR method,which increased the efficiency of homologous recombination,the sRNA RyhB1(108 bp) and RyhB2(106 bp) and the larger chromosome fragments 47-2 (10.4 kb),47-3(21.6 kb),47-3a (9.2 kb) and 47-3b (6.1 kb) were successfully deleted.[Conclusion] The two step PCR mutation technique was a simple and efficient method for the precise modification of sRNA and large fragment chromosome of Yersinia pestis.This method was suitable for gene knockout of whole genome of Yersinia pestis,which provided a powerful tool for gene expression and regulation,pathogenicity and virulence study of Yersinia pestis.