In phylogenetic，the algorithm of constructing evolutionary tree is the basis of studying evolutionary rela- tionship． The feasibility of constructing evolutionary tree based on k-mer dissimilarity algorithm d2S and adjacency method NJ was discussed and verified． The evolutionary tree was constructed for 12 16S rRNA and 36 bacterio- phage molecule sequences in two evolutionary scenarios (k =4，6，8，10)． Three types of 16S rRNA were clear- ly classified according to archaea，bacteria and eukaryotes，and the results are basically consistent with Woese cata- logue． At the same time，four types of bacteriophages can be distinguished according to the original evolutionary relationship，and the influence of parameter k is small，so the classification effect is very good． This demonstrates the powerful function of the dissimilarity algorithm d2S． Using the dissimilarity algorithm d2S and the adjacency method (NJ) to construct the evolutionary tree will provide a new algorithm and idea for the phylogenetic relation- ship of the system．