Development and initial validation of quantitative real-time PCR (qPCR) assays for the three serotypes of Marek's Disease Virus (MDV) are described. Also described is the development of an internal control qPCR assay that detects the chicken α2(VI) collagen gene. To reduce costs, a duplex assay for MDV1 and the internal control was also developed. The MDV qPCR assays were specific to their target gene and more sensitive than standard PCR when compared using Australian field and vaccine strains of MDV. All assays were found to have acceptable reproducibility. Relative abundance (RA) of MDVI and HVT viruses was quantified using the relative standard curve method in twenty experimentally infected chickens over a period of 7-35 days post infection and was shown to vary during the course of the infection. These qPCR assays will be useful for reliable differentiation and quantitation of MDV for a variety of applications.