Abstract: In this study, partial sequence of Riemerella anatipestifer 16S rRNA gene was amplified by polymerase chain reaction(PCR) using the bacteria 16S rRNA gene universal primers, which its length was 1 512 basepair (bp) and it has been recorded in GenBank and named as DQ078779. The BLAST analysis indicated that DQ078779 was the most similarity with reported 16S rRNA gene of R. anatipestifer. Moreover, speciesspecific primers were designed according to BLAST results of different 16S rRNA gene sequences of R. anatipestifer and E. coli, which 354 and 718 bp specific DNA fragment could be amplified by the two pairs of primers from R. anatipestifer and E. coli, respectively. Finally, dulex PCR with the specific primer for differentiating R. anatipestifer and E. coli infection in ducks was established, and genomic DNA or cultured bacteria or boiled liver tissue of ducks infected by R. anatipestifer and E. coli could be used as template.