The Mediterranean population of the loggerhead turtle (Caretta caretta) originates from a few colonization events from rookeries on Oceanic beaches. Mediterranean loggerhead turtles may have unique genetic adaptations to the region climatic conditions, due to their temperature sensitivity, affecting various biological functions. We used complete mitochondrial DNA sequences from 61 independent individuals sampled in the Mediterranean to infer the protein-coding variants. The 3D structures of the subset of proteins affected by non-synonymous substitutions were reconstructed to hypothesize the ensuing effects for the protein functionality from a structural and energetic point of view. By performing two consecutive sets of comparisons between proteins encoded by Hg IB vs. basal Hg II and Hg IB vs. derived Hg II we gained insights on whether the new variants replicate and potentiate the evolutionary trend observed in the long-term divergence between Hg IB and Hg II. Minor changes in protein stability were predicted in the Hg IB vs Hg II comparison, consistent with the long-term evolutionary viability of the amino acid substitutions in the two lineages. The five comparisons involving new variants, derived within Hg II, predicted a slight destabilization of the corresponding protein structure within the mitochondrial membrane 3D context, while drastic effects on the proteins’ functionality could be ruled out. Our analysis provides a novel view of the evolutionary dynamics of mitochondrial DNA and the potential functional implications of specific mutations associated with the colonization of the Mediterranean, contributing to a deeper understanding of the genetic diversity within and among C. caretta haplogroups.