The endangered fountain darter Etheostoma fonticola is found only in the Comal and San Marcos rivers in the Guadalupe River basin in central Texas, USA. Comal River fountain darters were believed to be extir- pated following a severe drought in the 1950s and were reintroduced in the early 1970s using 457 darters from the San Marcos River. In this study we used 23 microsatellite loci to describe and evaluate the genetic diversity, population structure and effective population size (Ne) of fountain darters. We also evaluated the genetic effect of the Comal River reintroduction and the influence of low-head dams (dams) on dispersal in both rivers. Bayesian analysis of individual genotypes and Analysis of Molecular Variation supported two distinct populations concordant with the two rivers. Estimates of Ne were much smaller (<10 %) than census size (Nc) in both rivers but did not indicate the populations are at risk of an immediate and rapid loss of genetic diversity. Coalescent-based estimates of the genetically effective number of founders (Nf) for the Comal River averaged about 49 darters and, together with the indices of genetic diversity and the bottleneck test (heterozygosity excess) results, were consistent with a founder event following the reintroduction in the Comal River. Finally, our results regarding the influence of dams on fountain darter dispersal were equivocal and did not support a conclusion. We recommend this issue be examined further as part of the fountain darter recovery program.