The evolutionary potential of a species directly impacts its ability to survive in fluctuating environments. A fundamental goal in wildlife conservation is enhancing this potential since anthropogenic pressures and rapid climate change are shifting environments at an alarming rate. One way to increase a species’ adaptive potential is through the delineation of management units based on population genomic analyses. Such units consist of evolutionarily significant gene pools requiring immediate conservation action. The delineation of priority units is fundamental for species on the brink of extinction, a predicament shared by numerous killifish species worldwide which face human-driven habitat transformations leading to the destruction of functioning ecosystems. The Mediterranean coast of the Iberian Peninsula has undergone one of the greatest human-driven habitat transformations in Europe since the turn of the 20th century as a result of agricultural exploitation and urbanization, imposing novel environmental pressures on various aquatic organisms including killifish species, such as the endangered and endemic Spanish toothcarp (Aphanius iberus Valenciennes, 1846). In the present study, we performed a SNP-based genetic analysis to delineate management units, or Operational Conservation Units (OCUs) for A. iberus, sampling a total of 176 individuals from 18 sample locations and analyzing their genetic structure, diversity, levels of gene flow, and degrees of genetic differentiation. Overall, the populations were highly structured with low genetic diversity values. Little to no gene flow was detected and FST values were high, indicating a large degree of genetic differentiation between populations, most likely attributable to habitat fragmentation. The results of our genetic analyses suggested the recommendation of nine OCUs for A. iberus, which should be implemented immediately into recovery programs to enhance the conservation management of this species. Using A. iberus as a study model, our research exemplifies how to delineate conservation priorities pertinent to killifish species with limited dispersal opportunity as a result of disruptions in population connectivity.