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Background: Vast migrations and subsequent assimilation processes have shaped the genetic composition of Southeast Asia, an area of close contact between several major ethnic groups. To better characterize the genetic variation of this region, we analyzed the entire mtDNA control region of 214 unrelated donors from Laos according to highest forensic quality standards. To detail the phylogeny, we inspected selected SNPs from the mtDNA coding region. For a posteriori data quality control, quasi-median network constructions and autosomal STR typing were performed. In order to describe the mtDNA setup of Laos more thoroughly, the data were subjected to population genetic comparisons with 16 East Asian groups., Results: The Laos sample exhibited ample mtDNA diversity, reflecting the huge number of ethnic groups listed. We found several new, so far undescribed mtDNA lineages in this dataset and surrounding populations. The Laos population was characteristic in terms of haplotype composition and genetic structure, however, genetic comparisons with other Southeast Asian populations revealed limited, but significant genetic differentiation. Notable differences in the maternal relationship to the major indigenous Southeast Asian ethnolinguistic groups were detected., Conclusions: In this study, we portray the great mtDNA variety of Laos for the first time. Our findings will contribute to clarify the migration history of the region. They encourage setting up regional and subpopulation databases, especially for forensic applications. The Laotian sequences will be incorporated into the collaborative EMPOP mtDNA database http://www.empop.org upon publication and will be available as the first mtDNA reference data for this country.