We studied the population genetic structure of
Cobitis vettonica
, an endangered freshwater fish species endemic to the Iberian Peninsula, in order to propose a biogeographic model of the responses of species to the multiple changes that occurred in the Iberian hydrological system during the Quaternary period. We also deciphered the relationship of
C
.
vettonica
with its sister species
C
.
paludica
, particularly in sympatric areas, and provide genetic information for conservation purposes. To achieve this goal, we analyzed both mitochondrial and nuclear data (the
cytochrome b
and the
nuclear recombination activating 1
genes) and a battery of single‐nucleotide polymorphisms (SNPs) of 248 individuals of
C
.
vettonica
or
C
.
paludica
from 38 localities, including some sympatric ones, covering the entire distribution area of
C
.
vettonica
. We highlight the important role played by the hydrogeomorphological processes and climatic changes that occurred in the Iberian Peninsula during the Quaternary on both the population structure of
C
.
vettonica
and its relationship with its sister species
C
.
paludica
. Our results support the genetic introgression of populations at the eastern limit of the distribution of
C
.
vettonica
. Furthermore, we postulate genetic introgression in sympatric areas. Finally, we propose the establishment or expansion of four Operational Conservation Units (OCUs) for
C
.
vettonica
, and highlight the threat faced by its populations due to the low level of genetic diversity detected for some of its populations and genetic introgression with
C
.
paludica
, which could eventually displace
C
.
vettonica
, resulting in a loss of diversity in this species.