For mitochondrial phylogenetic analysis, the best result comes from complete sequences. We therefore decided to sequence the entire mitochondrial DNA (mtDNA) (coding and D-loop regions) of 63 individuals selected in 3 small Ogliastra villages, an isolated area of eastern Sardinia: Talana, Urzulei, and Perdasdefogu. We studied at least one individual for each of the most frequent maternal genealogical lineages belonging to haplogroups H, V, J, K, T, U, and X. We found in our 63 samples, 172 and 69 sequence changes in the coding and in the D-loop region, respectively. Thirteen out of 172 sequence changes in the coding region are novel. It is our hypothesis that some of them are characteristic of the Ogliastra region and/or Sardinia. We reconstructed the phylogenetic network of the 63 complete mtDNA sequences for the 3 villages. We also drew a network including a large number of European sequences and calculated various indices of genetic diversity in Ogliastra. It appears that these small populations remained extremely isolated and genetically differentiated compared with other European populations. We also identified in our samples a never previously described subhaplogroup, U5b3, which seems peculiar to the Ogliastra region.