Phylogenetic analysis of 35 strains of Bacillus thuringiensis was performed based on PCR amplified fragment polymorphisms of flagellin genes (PCR-AFPF). From coefficient matrix, the genetic distance coefficient D among them ranged from 0.14286 to 0.81818. Ten clusters were divided by the taxonomic threshold of 0.42 from the UPGMA dendrogram, demonstrating that the genetic dissimilarities existed among 35 strains of B. thuringiensis corresponding to 35 different serovars. This method could provide a fast, convenient and accurate way to classify all subspecies of B. thuringiensis including strains that can not be classified using the H-antigen method.