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Black rats are of outstanding interest in parasitology and infective disease analysis. We used chromosome paints from both the mouse (Mus musculus) and the Norway rat (Rattus norvegicus) to characterize the genome of two Black rat subspecies from Italy. Both subspecies have two large metacentrics (n. 1, 4) not present in the Norway rat (2n = 42). Rattus rattus rattus has a diploid number of 2n = 38, while Rattus rattus frugivorous has two small metacentric “supernumerary” or B chromosomes for a diploid number of 2n = 38 + 2B. The 21 mouse paints gave 38 signals on the R. r. rattus karyotype and 39 signals in the R. r. frugivorous karyotype. The two metacentrics, not present in R. norvegicus, were hybridized by mouse 16/1/17 and mouse 4/10/15. These chromosomes are homologous to: RRA1 = RNO 5/7, and RRA4 = RNO 9/11 and not “4/7” and “11/12” as previously reported. Furthermore, the synteny of Chr 13 of the R. r. frugivorous with R. norvegicus Chr 16 and mouse Chrs 8/14 is not complete, because there is a small pericentromeric insertion of RNO Chr 18 (mouse Chr 18). If we consider only the two metacentrics, RRA1 and RRA4, the principal differences between R. norvegicus and R. rattus, then we can propose the derived synteny of 124 genes in the black rat. A comparison of the Z index between rats and mice shows an acceleration of genomic evolution among genus, species, and subspecies. The chromosomal differences between R. r. rattus x R. r. frugivorous suggest that they may be classified as different species because hybrids would produce 50% unbalanced gametes.