The European anchovy, Engraulis encrasicolus, is currently one of the principal target species for commercial fisheries in Europe. In this study, the mitochondrial Control Region (CR) and the Cytochrome Oxidase I (COI) mitochondrial gene were analyzed in 74 specimens of E. encrasicolus from four localities in the central Mediterranean. In both populations, the two markers revealed the presence of two main haplogroups, A and B, already detected in previous investigations of different classes of molecular markers. Both CR and COI markers consistently identified two haplogroups. The COI sequence analysis identified a non-synonymous transversion (T to G) at position 116 of the translated sequence, resulting in an amino acid change. All COI sequences of haplogroup A had an amino acid sequence with alanine in this position, while serine was present in the same position in haplogroup B. The two haplogroups A and B were also discriminated by the variable number of TACA elements at the 5'-end of the mitochondrial CR. The selection tests applied to the COI dataset revealed that codon 116 was not under positive selection, that seven amino acid changes were under purifying selection, and that two amino acids were under episodic positive selection.