Melanopsis etrusca is a local endemic species in the Melanopsidae, which is restricted to few thermal springs or their effluent rivulets or canals in a small region of the provinces Livorno and Grosseto in Tuscany, Italy. Historically M. etrusca has been reported from 11, possibly 12 populations, half of which have already gone extinct (EX, 4 populations during the twentieth century and another 2 in the last years) as a consequence of overexploitation of thermal springs for wellness uses (spas) in combination with water drainage and contamination caused by agricultural and mining activities. In the face of this considerable loss of populations we carried out a first genetic assessment of the remaining populations in order to get insights into population differentiation and population structure using mitochondrial as well as multilocus AFLP data. Our data suggest a clear differentiation into a western, a central and an eastern group of populations, which is largely consistent with a pattern of isolation-by-distance. Any reintroduction program should therefore be carefully planned according to the following criteria: (1) priority should be given to restocking actions that use specimens collected in the same stream, (2) cross-introduction programs should be considered only after the causes of decline have been removed/ceased and the whole resident population has gone EX, (3) the choice of specimens to be used for reintroduction programs should consider the adaptive (behavioural and physiological) and genetic affinity between source and EX populations.