Biogeography and the occurrence of small mammals are usually hard to investigate due to the small size and secretive habits of these mammals. Available data are particularly insufficient on minor islands and at national borders, where research efforts are usually scarce. Here we briefly updated the knowledge on murid rodents on two remote Italian small islands (Lampedusa and Pantelleria) at the southernmost Italian borders. During summer 2019, house mice and rats were sampled in Lampedusa and Pantelleria and molecular markers were sequenced for species identification. The new sequences of Mus domesticus were also compared with samples from previous works collected in Lampedusa, Pantelleria, and several localities in the Mediterranean basin. Moreover, our analyses provided the first genetic evidence of the occurrence of Rattus norvegicus in Lampedusa. To conclude, R. rattus was confirmed to be present in Pantelleria. The newly collected haplotype of M. domesticus from Pantelleria is similar to those currently known for Sicily, whereas the new haplotype from Lampedusa partially diverges from the ones previously described, and clusters with haplotypes from North Africa and the Middle East.