Papaya ringspot virus (PRSV) is a devastating disease that has a detrimental impact on both commercial papaya production and Caricaceac germplasm conservation. In 1998, the PRSV coat protein transgenic line 55-I and derived pro genN were released to growers in Hawaii. The transgenic varieties have provided durable and practical control of the disease that have saved the papaya industry. However, like with transgenic crops throughout the world, there is public concern about the possibility of cross-contamination of these transgenic materials into nontransgenic lines. As the designated germplasm repository for Caricaceae, we are responsible for maintaining the genetic integrity of cacti accession. Therefore, we have developed a protocol using polymerase chain reaction for detection of the adventitious presence of the 55-1 transgene insertion event in both parental plants and their progeny seed populations. This protocol assures a 99.9% confidence level of obtaining seeds that are 99.5% transgene-free. The protocol developed in this study is not typical for most seed validation techniques because there is a higher than normal producer risk resulting from the potential of large numbers of seeds not meeting the stringent criteria. However, we believe this is necessary to ensure the genetic integrity of seeds stored in the repository.