The Cucumber mosaic virus coat protein (CMV-CP) gene-transgenic pepper lines exhibit high tolerance to Cucumber mosaic virus (CMV) strains. In this study, E7, one of the CMVP0-CP transgenic chili pepper events selected by screening was further characterized. Southern blotting and inverse PCR analysis revealed that the E7 event contains a single copy of the inserted gene cassette whose flanking sequences appear to be noncoding and intergenic. We searched for pepper-specific DNA sequence candidates as an endogenous reference gene for GM-pepper detection. We found that only one copy of CaSIG4 and lipocalin genes are present in the pepper genome and their sequences were determined to be pepper-specific. The characterization of the genomic sequences flanking the transgene, as well as the availability of the pepper-specific single copy CaSIG4 and lipocalin genes as endogenous reference genes, enabled the design of E7-event-specific PCR-based quantitative detection methods. The CMV-CP protein levels in the CMV-inoculated wild-type pepper tissues were approximately 60 times higher than those in the uninoculated and CMV-inoculated E7 pepper tissues. These results suggested that the amount of CMV-CP expressed in transgenic pepper tissue was negligible relative to the amount of CMV-CP in the virus-infected wild-type pepper consumed by human beings. This work may prove useful for risk assessment studies of transgenic pepper lines. Furthermore, the characterized single copy genes, lipocalin and CaSIG4, may be used to develop a method to detect gene copy number variations in the pepper genome.