The present study was undertaken to quantify the Marek's Disease Virus (MDV) serotypes in vaccinated commercial layer flocks at 7, 14, 21, 28, 35 and 60-90 days post vaccination (dpv) and to correlate the pathogenic Gallid herpesvirus 2 (GaHV-2, MDV1) load with vaccine viral load of Gallid herpesvirus 3 (GaHV-3, MDV2) and Meleagridis herpesvirus 1 (MeHV-1, MDV3). A total of 25 commercial layer flocks were selected in and around Namakkal district of Tamil nadu, India and the feather pulp (FP) and blood samples were collected. Out of 25 flocks, 14 were revaccinated with bivalent vaccine, six were revaccinated with monovalent vaccine apart from the initial bivalent vaccination done at hatchery and five flocks were not revaccinated. SYBR green based real time PCR was used for absolute quantification of MDV serotypes. The pathogenic MDV1 load had shown an increasing trend until 21 dpv followed by a dip and again had shown a constant uptick between 60 and 90 dpv in the flocks that went on to develop MD outbreak. The flocks which had not encountered any Marek's Disease outbreak had shown increasing trend of MDV2 and 3 load until 21 dpv followed by a slight decrease but maintained a higher load when compared to MDV 1 which had marked a sharp decline between 60 and 90 dpv. Outbreak of MD was observed in seven (28%) out of 25 flocks between 18 and 27 weeks of age. It includes, two out of fourteen farms (14%) revaccinated with bivalent vaccine, two out of six farms (33%) revaccinated with MDV3 vaccine and three out of five farms (60%) without revaccination. The overall mean of vaccine viral load at various stages of dpv was constantly low where as pathogenic MDV 1 load was constantly high between 60 and 90 dpv in the flocks that went on to develop Marek's Disease during later part of life.