Objective In order to improve the discrimination power of mitochondrial DNA in forensic DNA typing, primer pairs which covered the mtDNA control region were designed. The methods of both amplification and DNA sequencing with new primers were established. Polymorphisms of mtDNA control region in 5 ethnic groups in China were analyzed using the established methods to provide a basic database. Molecular evolution and phylogenetic issues were discussed based on the mtDNA database to understand genetic relationship of different ethnic groups in China. Methods The primers were design according to the Anderson's sequence. The amplification system was optimized so that the PCR with different primers can be carried out under the same condition. The PCR products were sequenced usingSanger's terminator and fluorescent label techniques. Sequences were analyzed and compared base on Sequencing Analysis3.4 and Seq/Ede software. Validation study of forensic science was carried out using forensic samples such as hair, nail and badly degrade bone. The method has also been used in case work to validate the practicability. MAGA2 software was employed to analyze the genetic distance between samples and construct the phylogenentic tree. Result A total of five primer pairs was designed to cover the whole mtDNA control region and the neighbor part. The length of amplicon was from 299 bp to 452bp with different primer pairs. The successful result was obtained even if the DNA template was small to 0.015ng. The mtDNA typing of hair, nail and bone can be carried out with higher successful rate. Sequence analysis revealed that the number of variation site was 269, 316, 141, 117, 159 in Han, Li, Wei, Yao, Zang ethnic group, respectively, while the mean number of base pair with difference was 15.3, 15.9, 12.0, 10.5, 11.3 in Han, Li, Wei, Yao, Zang ethnic group, respectively. The genetic diversity of population samples from Han, Li, Wei, Yao, Zang ethnic group was 1,1, 0.9967, 0.9936, 1, respectively. The result of analysis for discrimination power from three hypervariable regions showed I higher than II and II higher than III. The third hypervariable region was different among the ethnic groups we studied. The analysis of variation in Han group revealed geographic difference. The genetic distances based on the sequence of mtDNA haplotypehave been obtained and the phylogenetic trees were constructed. Conclusion The mtDNA sequencing system using new primers was useful for forensic purpose. For difficult samples, DNA typing with this system was especially available and practical, and has been validated in criminal cases. After extending the sequenced region tb whole control region, the sequence in Han, Li, Wei, Yao, Zang ethnic groups revealed that the discrimination power of mtDNA was increased greatly. The novel STR and SNP loci found in the region of mtDNA also provide useful markers for screening test of forensic samples. The different polymorphisms among ethnic groups imply that it is necessary to establish reference database based on each ethnic group. The study of genetic relationship showed that the selected samples and the sequenced mtDNA region satisfied the request of the genetic analysis. The novel variable site provided some index to establish a perfect phylogenetic tree.