The entire genome of Cydia pomonella granulovirus (CpGV) was systematically screened for origins of DNA replication, using an infection-dependent DNA replication assay in the granulovirus-permissive Cydia pomonella cell line, Cp14R. All seven cosmids in an overlapping library that covered the CpGV genome were found to replicate in the assay. A genomic library of 32 overlapping plasmids was subsequently screened. Plasmids that replicated were in turn subcloned into 1-2 kbp overlapping fragments. Eleven subclones replicated, each containing at least one of the 13 single-copy 74-76 bp imperfect palindromes, previously identified in the CpGV genome as possible origins of replication. Genome fragments of 156 bp, each containing one of the 13 palindromes, were cloned to verify replication and provided confirmation that these 13 palindromes are the only origins of replication in the genome. A real-time PCR method was developed for the quantification of DNA replication, which eliminated the need for Southern blotting and hybridization. A set of deletion clones allowed further quantitative characterization of one of the palindromes. The previously proposed non-homologous region origin of replication did not replicate in the assay.