The Azores islands (Portugal), uninhabited when discovered by Portuguese navigators in the fifteenth century, are located in the Atlantic Ocean 1500 km from the European mainland. The archipelago is formed by nine islands of volcanic origin that define three geographical groups: Eastern (S. Miguel and Sta. Maria), Central (Terceira, Faial, Pico, Graciosa and S. Jorge) and Western (Flores and Corvo). To improve the genetic characterisation of the Azorean population, and to clarify some aspects related to the history of settlement, a study of mtDNA was conducted in the population of the archipelago. The HVRI region was sequenced and specific RFLPs were screened in 146 samples obtained from unrelated individuals with Azorean ancestry (50 from the Eastern group, 60 from the Central group, and 37 from the Western group). Samples were classified into haplogroups based on the information obtained from both sequencing and RFLP analysis. All the analyses performed support the idea that, in the whole group of islands, the majority of mtDNA lineages originated from the Iberian Peninsula, mainly from Portugal (mainland). However contributions from other European populations, especially from Northern Europe, cannot be disregarded. The values obtained for the various diversity parameters in the Azores archipelago indicate that the Azorean population, as a whole, does not exhibit the typical characteristics of an isolated population. The analysis of genetic data by groups of islands showed that the Western group exhibited particular features. The distribution of haplogroups in the Western group is very atypical, being significantly different from what is observed in the Eastern and Central groups. Furthermore, the diversity values are, in general, lower than those observed in other populations used for comparison. African haplogroups were found in all the groups of islands. Therefore the presence of Moorish and African slaves on the islands, as reported in historical sources, is supported by the mtDNA genetic data, especially in the Eastern group. The presence of Jews in the Central group is also supported by the mtDNA data. Neither historical nor genetic data (phylogeography of mtDNA) supports the idea of a differential settlement history for the Western group; however, it is represented in the phylogenies as an isolated branch. The effect of genetic drift, induced by the reduced population size since peopling occurred, has led to a very atypical distribution of haplogroups/haplotypes in this group of islands. We cannot ignore the influence of biodemographic and genetic processes, namely founder effect, genetic drift, migration, and even recent mutational events in the mtDNA lineages of the Azorean populations. Nevertheless, a great part of the variation in the Azorean mtDNA can be explained by the settlement history.