Due to their basal position in the vertebrate phylogenetic tree, the study on elasmobranch genetics and cytogenetics can provide remarkable information on the mechanisms underlying the evolution of all vertebrates. In recent years, different molecular approaches have been used to study the relationships between the different taxonomic groups of cartilaginous fish, among them are the physical mapping of specific nucleotide sequences on chromosomes. However, these are controversial, particularly in Torpediniformes in which the species have different karyological parameters. The purpose of this paper is to gather the molecular markers so far present in literature that were used to reconstruct the phylogenetic position of Torpediniformes with respect to the other Batoidea and to discriminate between the various chromosome pairs in the endemic species in the Mediterranean Sea, Torpedo torpedo, T. marmorata and T. nobiliana. The 5S and 18S ribosomal DNA, the HpaI and Alu SINE, the telomeric (TTAGGG)n and the spermatogenesis-related SPATA 16, SPATA 18, and UTY sequences were particularly useful. These last genomic segments were also able to differentiate between the male and the female karyotypes. Moreover, the torpedoes showed a particular genomic organization, especially Torpedo torpedo, in which large quantities of highly repeated DNA and a characteristic distribution of heterochromatin, which is never centromeric, were observed.