The risk of gene escape is a major concern when transgenic crops are released into the environment, especially for crops that are outcrossing, spread vegetatively, and produce fruit dispersed by animals. However, gene flow from crops with these traits can be difficult to quantify. One such crop, strawberry (Fragaria x ananassa), is cultivated in the southeastern USA, often near populations of the native species Fragaria virginiana, which can cross with the crop. In the present study, we examined molecular marker variation in Fragaria species, identified markers that are present in cultivated strawberry and absent/rare in wild F. virginiana populations isolated from strawberry farms, and used 24 of the 'cultivar markers' to estimate rates of past and present gene flow from cultivated strawberry to five F. virginiana populations located 100 m to 7.5 km from strawberry crop fields. Eighty percent of the cultivar markers were present in the populations, including markers unique to modern cultivars and markers unique to cultivars no longer grown in the region. Within populations, the plants had different combinations of markers. In three populations, plants were identified that had markers derived from more than one cultivar. The markers were present at widely varying frequencies, suggesting differences in persistence. These results are consistent with past and present gene flow, from strawberry cultivars to F. virginiana and among hybrid and nonhybrid plants.