We designed one multiplex assay with a reduced number of SNPs from whole mitochondrial genome as a screening approach for forensic purposes and developed a multiplex SNaPshot assay with 26 mitochondrial SNPs (mtSNPs). This assay included 16 target mtSNPs that defined the main haplogroups in Chinese population and ten hot-spot mtSNPs found by pyrosequencing. To validate our multiplex mtSNP assay, we not only analyzed a Chinese Han population sample, but also sequenced the complete control region of same set of individuals. Fifty-one haplotypes were observed in 204 individuals using our multiplex mtSNP assay and the haplotype diversity was estimated to be 0.9626. Our multiplex mtSNP assay could also distinguish some individuals sharing the same control region sequences. The same mtSNP profiles were obtained from the bloodstain, hair shaft, and salivary swab from same individuals. A good profile could be obtained with 50 pg of DNA. It was evident that our multiplex mtSNP assay not only improved the discrimination power, but also allowed allocating mitochondrial DNA profiles to particular haplogroups not clearly defined with the control region alone. We highlight the importance of the balance of target mtSNPs for haplogroup assignment and hot-spot mtSNPs for increasing discrimination power.