The population structure of the land snail Ventridens ligera (Say, 1821) was investigated in the Potomac River Basin (Virginia, Maryland, U.S.A.). Animals were collected from two islands and the adjacent riverbanks along an 8.8-km stretch of the river. Four landlocked populations in Illinois and Maryland were also sampled to provide a comparison to the river populations. A total of 246 individuals were genotyped with five newly designed species-specific microsatellite primers. Low pairwise F_ST values (<0.0477) among the Potomac River sites suggest high levels of gene flow between the populations. In contrast, the landlocked populations had high F_ST values (0.0738 to 0.6004) which suggest genetic structuring, most likely due to physical isolation, because F_ST values >0.2 indicate population structuring. Low-level isolation by distance was found among the Potomac River populations, and the low F_ST suggests that the river is facilitating gene flow rather than acting as a barrier.