Forensic analysis of mitochondrial displacement loop (D-loop) sequences using Sanger sequencing or SNP detection by minisequencing is well established Pyrosequencing has become an important alternative because it enables high-through put analysis and the quantification of individual mitochondrial DNAs (mtDNAs) in samples originating from more than one individual. DNA typing of the mitochondrial D-loop region is usually the method of choice if STR analysis fails because of trace amounts of DNA and/or extensive degradation. The main aim of the present work was to optimize tie efficiency of pyro-sequencing. To do this, 31 SNPs within the hypervariable regions I and II of the D-loop of human mtDNA were simultaneously analyzed. As a novel approach, we applied two sets of amplification primers for the multiplexing assay. These went in combination with four sequencing primers for pyrosequencing. This method was compared with conventional sequencing of mtDNA from blood and biological trade materials.