Objective To establish a rapid, reliable method for identifying and typing of Clostridium botulinum using 16S rDNA sequencing and phylogenetic tree construction. Methods Clostridium genome templates were extracted from 9 strains, including LCL063, 830110, LC175, LCL155, 66418, N153, 61082, ALASKA, and IWAN AI; 16S rRNA genes were amplified by PCR with the 16S rRNA specific primers, and then the PCR products were cloned to pGEM-T Easy vector and sequenced. Finally, the sequence of 16S rDNA was analyzed by Clustal and Mega program; the Phylogenetic trees were constructed by Neighor joining and Maximum parsimony. Results It was found that LCL063, 830110, and LCL175 were BONT/E producing Clostridium butyricums; IWANAI and ALASKA were Clostridium botulccum type E. The results of the present method were consistent with those of the conventional method. Conclusion 16S rRNA sequencing combined with phylogenetic tree analysis a rapid and accurate method in Clostriduum bouulccum identification, and the method may serve as a criterion for bacterial typing with the completion of ribosomal RNA data bank.