Technical Abstract: Papaya Ringspot Virus (PRSV) is a devastating disease that has a detrimental impact on both commercial papaya production and Caricaceae germplasm conservation. The PRSV coat protein transgenic line 55-1 and derived progeny are resistant to PRSV and have saved the papaya industry in Hawaii. However, similar to transgenic crops throughout the world there is public concern on cross contamination of non-transgenic lines. As the designated germplasm repository for Caricaceae we are responsible for maintaining the genetic integrity of each accession. Therefore, we have developed a protocol utilizing polymerase chain reaction (PCR) for the detection of adventitious presence of transgenic material in both the parental plants and the resulting seed population by testing each for the 55-1 transformation event to assure a 99.9% chance of obtaining greater than 99.5% transgene free seeds. The protocol developed in this study is not typical for most seed validation techniques since this there is a higher than normal producer risk in testing however, we believe this is necessary to ensure the genetic integrity of seeds stored in the repository.