First genetic analysis from triple burial of Dolni Vestonice. The human remains from the Dolni Vestonice site are among the oldest and best preserved early modern human fossils (Homo sapiens) known to date. In particular the enigmatic triple burial presented by the three individuals CV13-15 and their potential ritual positioning are widely discussed. In order to determine the potential genetic relationship between those three human skeletons we carried out an ancient DNA analysis. Using new high-throughput DNA sequencing technologies we were able to obtain a complete and two partial mitochondrial genome DNAs (mtDNA) that are passed on from the mother to their offspring. The DNA retrieved from the three skeletons shows aging patterns typical for Pleistocene DNA suggesting that the DNA is indeed ancient and not derived from recent contamination of modern humans handling the specimens. All three individuals carry a different mtDNA sequence and are therefore not directly maternally related, i.e. no siblings or cousins. The three mtDNAs are similar in their sequence to previously determined DNA from Pleistocene and early Holocene hunter gatherers. Our results show that ancient DNA is preserved in all three studied Dolni Vestonice human fossils, the mtDNA suggests no maternal relationship among them and shows sequences typical for Pleistocene modern humans in Europe. Thus the Dolni Vestonice site is one of the best sites to study and understand the genetic relationships within Pleistocene modern human population.