Oak wilt caused by Ceratocystis fagacearum is a significant disease of Quercus spp. in the eastern United States. Early and accurate detection of the pathogen is particularly important when disease control is planned. Nested and real-time polymerase chain reaction (PCR) methods utilizing fungal DNA extracted from sapwood drill shavings of red, bur, and white oak at different stages of disease development were compared with culture-based detection from sapwood. The pathogen was detected in all (n = 3) actively wilting branches of each of nine red oak trees using all three methods. The lowest detection rate (33% of assayed branches; 6 of 8 trees) for actively wilting branches was found for white oak using isolation while nested PCR had a branch detection rate of 100% (8 of 8 trees) and real-time PCR of 87% (8 of 8 trees) for the same samples. For both bur and white oak, the pathogen was not detected by isolation in branches over 1 year after their death but was detected using both PCR methods. Only the PCR assays detected the fungus in sapwood samples underlying remnants of sporulation mats (n = 21; 90%, nested and 62%, real-time) on red oak. These PCR methods offer several significant improvements for laboratory-based detection methods of C. fagacearum.