AbstractChloroplast genome diversity in cork oak (Quercus suber) is characterised by the occurrence of haplotypes that are akin to those found in other Mediterranean oak species, particularly in Q. ilex and Q. rotundifolia, suggesting the possible presence of an introgressed chloroplast lineage. To further investigate this pattern, we reconstructed chloroplast haplotypes by sequencing four chloroplast markers (cpDNA), sampled across 181 individuals and 10 taxa. Our analyses resulted in the identification of two diversified chloroplast haplogroups in Q. suber, corresponding to a geographically widespread lineage and an Afro-Iberian lineage. Time-calibrated phylogenetic analyses of cpDNA point to a Miocene origin of the two haplogroups in Q. suber, suggesting that the Afro-Iberian lineage was present in cork oak before the onset of glaciation periods. The persistence of the two haplogroups in the western part of the species distribution range may be a consequence of either ancient introgression events or chloroplast lineage sorting, combined with different fixation in refugia through glaciation periods. Our results provide a comprehensive insight on the origins of chloroplast diversity in these ecologically and economically important Mediterranean oaks.