Technical Abstract: Alfalfa (Medicago sativa L.) is a perennial species with an out-crossing rate of between 67 and 77 percent. Pollen is primarily vectored by bees. An accurate understanding of the frequency with which foreign pollen results in viable seed set is critical to establishing appropriate mechanisms for isolation and maintenance of genetic purity. Studies were conducted to determine the frequency of pollen mediated gene flow under commercial seed production conditions with either honey bees (Apis mellifera) or a combination of honey bees and alfalfa leaf cutter bees (Megachile rotundata) as pollinators. A 318 acre commercial seed production field with the Roundup Ready (RR) trait conferring Roundup® herbicide tolerance was used as a pollen-marking tool. Study site/trap plots (1.8 acres) were established between 165 ft and 5 miles. Approximately 1.2 million seedlings were evaluated for the presence of the RR trait from these study sites during each of two production years. There was a logarithmic decrease in gene flow between 165 ft and 3 miles from the RR source field. Gene flow averaged 1.67% at 165 ft, 0.22 percent at 1 mile, and was not detectable at 5 miles. These results are being utilized by seed certification agencies to develop isolation distances, and mapping and stewardship programs to be employed during the production of seed for markets that are sensitive to the presence of transgenic traits.