As the major forest pest in China, Dendrolimus includes Dendrolimus punctatus Walker, D. tabulaeformis Tsai et Liu, D. spectabilis Butler, D. superans Butler, D. houi Lajonquiere and D. kikuchii Matsumura. During sequential outbreaks, economic damage was serious and the forest appeared to be burned. So a large number of studies were carried out at different levels but the research concerning the genetic variation and differentiation of populations based on a single specific gene mutation has not been reported. In order to clarify the genetic structure of the populations of Dendrolimus, and provide the scientific basis for prevention and treatment of Dendrolimus, a fragment with 387 bp of the mitochondrial cytochrome b (Cyt b) gene sequence in different populations of D. punctatus Walker, D. spectabilis Butler and D. tabulaeformis Tsai et Liu was amplified and sequenced. Nucleotide composition, transitions and transversions, amino acid composition, genetic distance, and the phylogenetic relationship were analyzed with molecular biology software. The results indicated that the average contents of A, T, C and G were 40.1, 33.5, 9.5 and 16.9%, respectively, and the contents of A T 73.6% were obviously higher than that of C G 26.4%. Cyt b exhibits an A/T bias across all sites which was the most prominent at the third position of codon with the highest content of 86.5% but only 1.1% of C at the same position. There was no significant difference for A/T bias in the populations. Nucleotide substitution occurred mostly at the third position. Transitions were greater than transversions, while substitutions of intraspecific populations were higher than interspecific populations. Thirty nine nucleotide sites and eleven amino acids showed mutation in this sequence fragment and the variability were 10.1% and 8.5%. Nucleotide sequences and amino acid sequences in genetic distance were 0.000-0.100 and 0. 000-0.086, indicating a low genetic variation. The amino acid sequences were believed to be more accurate than nucleotide sequences in genetic distance. Polypeptide is composed of nineteen amino acids except for glutamic acid and amino acid differences have little to do with populations. The most common amino acids and codons are lysine[AAA] and asparagine[AAU].The average content of AAA and AAU is 15.00% and 10.63%, respcetively. Cluster analysis showed that the genetic distance between D. punctatus Walker and D. tabulaeformis is relatively close, while genetic differentiation exists between D. punctatus Walker and D. spectabilis Butler. The population genetic differentiation was related to ecological environment. These results provided a basic molecular biology clue to the studies on population genetics and ecological control of Dendrolimus.