The distribution of the Galápagos land iguanas Conolophus subcristatus has been strongly affected by human activities in the last century. Previously widespread throughout the whole archipelago, today they inhabit only few islands, with populations often small and isolated. In this study, we analyzed the population genetic structure of land iguanas from Santa Cruz Island to investigate the genetic implications of a semi-captive conservation program that started in middle 1970s and is still ongoing. Nine microsatellites were used to measure the level of genetic variability and to detect potential evidence of inbreeding and genetic sub-structure. Furthermore, we used approximate Bayesian computation, together with software packages for coalescent-based simulations, to test a priori hypotheses in different demographic scenarios. Despite the abrupt reduction in size of the original population, no evidence of inbreeding was found, and the levels of genetic variability were similar to those of undisturbed populations of the archipelago. Nevertheless, the source and the repatriated populations started differentiating (FST=0.016) and genetic sub-structure was found. Following our results and the simulation of possible future scenarios, we suggest the genetic measures that should be adopted to avoid further genetic variability depletion and preserve this vulnerable endemic species.