The Basque population inhabits the Franco-Cantabrian region in southwest Europe where Palaeolithic human groups took refuge during the Last Glacial Maximum. Basques have been an isolated population, largely considered as one of the most ancient European populations and it is possible that they maintained some pre-Neolithic genetic characteristics. This work shows the results of mitochondrial DNA analysis of seven ancient human remains from the Cave of Santimamiñe in the Basque Country dated from Mesolithic to the Late Roman period. In addition, we compared these data with those obtained from a modern sample of Basque population, 158 individuals that nowadays inhabits next to the cave. The results support the hypothesis that Iberians might have been less affected by the Neolithic mitochondrial lineages carried from the Near East than populations of Central Europe and revealed the unexpected presence of prehistoric maternal lineages such as U5a2a and U3a in the Basque region. Comparison between ancient and current population samples upholds the hypothesis of continuity of the maternal lineages in the area of the Franco-Cantabrian region.