The white-clawed crayfish is endemic to western and southern Europe and its populations have decreased over recent decades. Spanish populations are generally poorly represented in scientific reports and are usually studied only with a single molecular marker. Here, we use two mitochondrial markers (cytochrome oxidase subunit I and rDNA 16S genes) to examine levels and patterns of genetic structure across the range of the species’ distribution in Spain. Data reveal the existence of two main genetic groups of white-clawed crayfish in Spain with the Ebro basin as a possible contact zone. Processes occurred in historical and recent times, such as genetic drift and translocations, contribute greatly to this genetic structure. Levels of genetic variability and genetic structure of Spanish populations together with demographic inferences suggest that the species established in the Iberian Peninsula, at least since the Late Pleistocene. Knowing the true origin of the Spanish populations is crucial when deciding upon the management policies that should be followed. Given the lack of any clear evidence against its indigenous status, we propose that current protection and conservation measures should be maintained. From a management point of view, we suggest that Spanish population should be considered as a single evolutionary significant unit (ESU) with two management units (MUs) corresponding with the genetic clusters detected in the present study.