Island radiations can offer challenging systems for the implementation of conservation policies because descendent populations may exhibit different levels of adaptive divergence, reproductive isolation, and phylogenetic distinctiveness. This seems particularly true for the endangered Galapagos giant tortoises (Geochelone nigra), which comprise a lineage that radiated rapidly and concomitantly with the evolution of the archipelago. We used mitochondrial DNA sequences and microsatellite markers to investigate the genetic structure, and to reconstruct genealogical relationships and the history of population colonization of giant tortoises from the Islands of Santa Cruz and Pinzon, including samples of a basal taxon from the Island of San Cristobal. Populations displayed marked genetic divergence, contrasting demographic histories, and deep phylogeographic structure. The pattern of diversification among populations was consistent with geological and biogeographic history, and to some extent, with adaptive and morphological divergence. Results strongly indicate the presence of a minimum of four conservation units with long-standing evolutionary separation: two in Santa Cruz, one in Pinzon, and one in San Cristobal. We propose that these findings be effectively integrated with other existing data by the appropriate environmental agencies to evaluate current conservation efforts and implement new strategies aimed at protecting the integrity and diversity of giant tortoise populations.