Boxwood dieback, caused by Colletotrichum theobromicola, is spreading at an alarming rate in the boxwood industry in the United States. Although C. theobromicola has been accepted as a distinct species within the C. gloeosporioides species complex, it is difficult or impossible to distinguish from other closely related species on the basis of morphology. Moreover, molecular identification requires amplification and sequencing of multiple loci, which can be expensive and time consuming. Because boxwood dieback produces above-ground symptoms similar to those produced by Phytophthora root rot, visual disease diagnosis may be inaccurate and result in costly and ineffective management decisions. Furthermore, previous pathogenicity reports have shown that the symptoms of boxwood dieback appear 10-12 weeks after inoculation under greenhouse conditions. This delay in the onset of disease symptoms may lead to infected boxwood liners being traded in the horticulture industry and unwitting dissemination of the causal agent. Therefore, an accurate and a rapid diagnostic tool is required to detect C. theobromicola in suspected liners at early growth stages. This will allow boxwood producers to implement effective disease management strategies, and prevent the spread of the disease. The main objective of this study is to develop a diagnostic taqman real-time PCR assay for early detection and quantification of C. theobromicola from boxwood.