Transgenic plants over-expressing virus coat protein genes have attracted particular interest from researchers recently, due primarily to their tolerance to viral infection. The transgenic watermelon rootstock analyzed in this study contains the introduced cucumber green mottle mosaic virus coat protein (CGMMV-CP) gene. The primary objective of this study was to determine the copy number, integration site, and expression level of the transgene element, in order to establish a scientific framework for the molecular genetic assessment of transgenic plant rootstocks. The results of our Southern blot analysis indicated that a single copy of the CGMMV-CP gene was inserted into the genome of a transgenic watermelon rootstock. We also identified the genomic sequences flanking the integration site of the transgene via inverse PCR analysis. In an effort to find a sequence usable as an internal positive control for the screening of the transgenic watermelons, we determined that the Sat gene appears as one copy within their genomes and is watermelon-specific. The information from the integrated site and the internal positive control sequence was utilized to establish a new event-specific PCR-based detection method. The expression of both CGMMV-CP mRNA and protein was detected in the transgenic watermelon rootstocks but not in watermelon scions, thereby suggesting that the tissues of the watermelon scions are free of the introduced gene products.