Italy hosts a large number of endemic freshwater fish species due to complex geological events which promoted genetic differentiation and allopatric speciation. Among them, the South European roach Sarmarutilus rubilio inhabits various freshwater environments in three different ichthyogeographic districts. We investigated the genetic diversity of S. rubilio using two different mitochondrial markers (COI and CR), aiming to define its relationship with other similar taxa from the Balkan area and, from a phylogeographic perspective, test the effects of past hydrogeological dynamics of Italian river basins on its genetic structure and demographic history. Our analysis highlighted a marked genetic divergence between S. rubilio and all other roach species and, among Italian samples, revealed the existence of three deeply divergent geographic haplogroups, named A, B and C. Haplogroup C likely corresponds to a new putative cryptic species and is located at the northern border of the South European roach range; haplogroup B is restricted to Southern Italy; and haplogroup A is widespread across the entire range and in some sites it is in co-occurrence with C or B. Their origin is probably related to the tectonic uplifting of the Apuan Alps in the north and of the Colli Albani Volcano in the south during the Pleistocene, which promoted isolation and vicariance followed by secondary contacts.