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Background/Aims: Present-day Iran has long represented a natural hub for the expansion of human genes and cultures. That being so, the overlapping of prehistoric and more recent demographic events interacting at different time scales with geographical and cultural barriers has yielded a tangled patchwork of anthropological types within this narrow area. This study aims to comprehensively evaluate this ethnic mosaic by depicting a fine-grained picture of the Iranian mitochondrial landscape. Methods: mtDNA variability at both HVS-I and coding regions was surveyed in 718 unrelated individuals belonging to 14 Iranian ethnic groups characterized by different languages, religions and patterns of subsistence. Results: A discordant pattern of high ethno-linguistic and low mtDNA heterogeneity was observed for the whole examined Iranian sample. Geographical factors and cultural/linguistic differences actually represented barriers to matrilineal gene flow only for the Baloch, Lur from Yasouj, Zoroastrian and Jewish groups, for which unusual reduced levels of mtDNA variability and high inter-population distances were found. Conclusion: Deep rooting genealogies and endogamy in a few of the examined ethnic groups might have preserved ancestral lineages that can be representative of Proto-Indo-Iranian or prehistoric mitochondrial profiles which survived relatively recent external contributions to the Iranian gene pool.