Population and conservation genetics of two freshwater fish species, Notropis heterodon and Notropis heterolepis, were evaluated in north-eastern Illinois, U.S.A., where both species have severely declined. Fishes were sampled from two remnant populations occurring in small glacial lakes (source samples) and from two man-made ponds that had been stocked with fishes from those same lakes (sanctuary samples). The goal was to obtain information that would help inform conservation programme planning to reintroduce sanctuary fishes to areas where both species are extirpated. Microsatellite data showed that the two species were genetically quite distinct and there was no evidence of hybridization in either source or sanctuary samples. Within each species, source and sanctuary samples had moderate levels of heterozygosity and were not significantly different from each other. Many alleles observed in the source samples, however, were not detected in the sanctuary samples, indicating that translocation had resulted in reduced allelic diversity of the sanctuary samples. Sibship analysis indicated that full and half sibs occurred within source-lake samples, thus reducing the effective population size of the reintroduced stock. Taken together, these results suggest that source-lake stocks rather than sanctuary stocks are more appropriate for future reintroductions of both species in their native range, unless sanctuary populations can be established with hundreds of fishes. Also, fishes should be harvested from multiple locations in source lakes to avoid over-representation of family groups.