The DNA sequence diversity of Sardina pilchardus (Walbaum, 1792) and some closely related species of Clupeomorpha was investigated using the mitochondrial DNA gene encoding cytochrome b. The nucleotide sequences of complete and partial mtDNA cytochrome b were determined in numerous specimens. Sequence divergence between species and genera was evenly distributed in the cytochrome b gene but rather high compared to reports for other fish species. Phylogenetic analyses on complete cytochrome b were used to study the relationships among the considered species. S. pilchardus was easily differentiated, showing a genetic distance of 0.25 with respect to Clupeidae species and 0.26 with respect to the other species. A species-specific short fragment (<150 bp) was isolated by polymerase chain reaction (PCR) using primers designed for Clupeomorpha. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with two restriction enzymes (MnlI/HinfI) was optimized for unambiguous differentiation of S. pilchardus from the other species tested (raw and canned products).