The white-clawed crayfish (Austropotamobius italicus), a cornerstone of Spain's aquatic ecosystems, was once widely distributed throughout much of the country. Unfortunately, its populations have suffered very strong declines over the last 40 years due to the spread of introduced species (red swamp and signal crayfishes), diseases, habitat loss and other anthropogenic impacts. The present work examines the genetic variation in 23 Spanish and four Italian populations of white-clawed crayfish via the analysis of microsatellite loci. The data show genetic variation in the Spanish populations to be affected by drastic and successive bottlenecks. Notwithstanding, the diversity of these Spanish populations in terms of observed heterozygosity is similar to or even higher than that recorded for other European populations studied using these same markers. North-central Spanish populations are clearly differentiated from the country's remaining populations; they should be considered distinct management units. Processes occurred in historical and recent times, such as genetic drift and translocations, contribute greatly to this genetic structure. These data provide useful information for conservation of this species, since the preservation of its population structure and genetic variability should be goals for management decisions.