The genetic composition of the medieval populations of Central Europe, Poland in particular, has been poorly investigated to date. Although a few DNA datasets from Poland have been published recently, no large-scale ancient DNA study on medieval populations has hitherto been reported. This paper reports the study of mitochondrial DNA (mtDNA) and presents the first population-level human DNA study from Lesser Poland by establishing mitochondrial DNA pro-files for 13 samples from the Grodowice cemetery dated to the Medieval Period (11th to mid-13th century). The medieval sequences encompass almost the entire range of Western Eurasian macro-haplogroups: H, J, U. Interestingly, there is one sample which belongs to the Asian haplogroup G. aDNA sequences were compared with a dataset of 35,203 present-day
sequences of the HVR I region of mtDNA including European, Near Eastern, and Asian populations, as well as 775 ancient sequences. Analyses of population genetics were performed, including genetic distances (Fst), multidimensional scaling (MDS), principal component analysis (PCA) and shared haplotype analysis (SHA). The shared haplotype analysis (SHA) showed that the medieval population from Grodowice shares the majority of haplotypes with the medieval populations from the contact-zones of today’s Slovakia and Croatia (53.85%) as well as with Hungarian conquerors (46.15%).