Since the reduced genetic diversity found in isolates should simplify the study of complex traits, analyses of patterns of homogeneity within populations are of particular interest. We analysed the mtDNA haplogroups and hypervariable segment I (HVS-I) sequences of 475 individuals from a geographically restricted and isolated area (Ogliastra) within Sardinia, comprehending 175 random samples from 20 out of 23 villages. The remaining 300 subjects were chosen from the other three villages, Talana, Urzulei and Perdasdefogu, by sampling all maternal lineages. A comparison with other European populations reveals that Ogliastra ranks among the most genetically homogenous population and that it has been small and isolated throughout its history. The lack of variation and the high genetic homogeneity indicate that an important founder event and a demographic expansion took place during the Neolithic (approximately 7700 years before present) in Ogliastra's mtDNA gene pool. We present highly resolved phylogenetic networks for Ogliastra and for the three sub-isolates. MtDNA differentiation in the sub-populations versus Ogliastra is revealed by a strong demarcation in their genetic pools due to distinctive founder effects and genetic drift. We found that genetic homogeneity strictly depends on a scale factor in population size and on sampling methodology. The outstanding homogeneity and the reduced female gene pool observed in Ogliastra, in the European context, hide an extremely marked differentiation in sub-isolates originated from the same archaic population. Although Ogliastra can be considered a genetically homogeneous isolate, small villages' divergent genetic histories underline the importance of more systematic analysis of DNA variation between and within populations.