Unique evolutionary potential could be lost when a population goes extinct or when individuals are translocated to other existing populations. Therefore, in order to identify priorities and to predict the efficiency and consequences of conservation actions, information is needed on the genetic structure of natural populations. In the urbanized and diverse landscapes of Flanders, Belgium, natterjack toad ( Epidalea calamita ) populations have been declining over the last decades. Therefore, this species is subjected to a wide range of different types of conservation measures (e.g. habitat management, corridor development, translocations). However, more information is needed on its genetic population structure. In this study, we sampled egg clutches from six populations and studied their genetic structure with six microsatellite markers. In total, 184 samples from 99 different egg strings were genotyped. Observed heterozygosity was generally high, even for the small and isolated populations (overall mean H O = ...