The European DNA Profiling (EDNAP) Group performed a collaborative exercise on a mitochondrial (mt) DNA screening assay that targeted 16 nucleotide positions in the coding region and allowed for the discrimination of major west Eurasian mtDNA haplogroups. The purpose of the exercise was to evaluate the stability and reproducibility of the self-developed multiplex-PCR and multiplex-single base extension kit by blind-testing saliva and hair shaft samples provided by the organizing laboratory. The overall success rate in obtaining useful results was high given that some of the participating laboratories had no previous experience with the technology and/or mtDNA analysis. The results of this collaborative exercise stimulate the expansion of screening methods in forensic laboratories to increase efficiency and performance of mtDNA typing, and thus demonstrates that mtDNA SNP typing is a powerful tool for forensic casework analysis.