Marek's disease is still a major problem among Egyptian poultry flocks, despite the intensive vaccination programs against the disease. This study was conducted to investigate the prevalence of MDV infection among forty-four flocks of breeders and layers with ages ranged from 4-20 months. Feather follicle specimens were collected during 2012-2015 from 44 vaccinated chicken flocks showing emaciation and visceral tumors. The samples were tested by PCR using three pairs of primers. Nineteen flocks were positive for MDV using primers targeting the UL 19 gene with a percentage of 43.2%. Three flocks were shown to be positive by primers targeting the meq and 132 bp tandem repeat genes with a percentage of 6.8%. Inoculation of duck embryo fibroblast (DEF) and chick embryo fibroblast (CEF) showed CPE in the form of plaques formation within 5-14 days post inoculation. Sequencing of meq and 132 tandem repeat genes of the 3 samples revealed that the isolated strains exhibited 99% homology with the very virulent European, Chinese, American, Indian and Egyptian MDV isolates. In conclusion, although the availability of MDV vaccines especially HVT vaccine which is used in the examined flocks in the present study, the disease was recorded. Thus, indicating that HVT vaccines are unable to protect completely against more virulent strains. Therefore, there is a need to develop a new strategy and types of vaccination to be able to protect against new strains of virus.