Marek’s disease virus (MDV) causes immunosuppression and tumors in poultry. Lymphoma induction in Marek’s disease (MD) has been associated with serotype-1. The study employed the use of two-step-PCR with primers directed to gB-gene of MDV to determine and differentiate MDV-serotypes associated with MD-outbreaks in poultry-flocks using tissue samples with neoplastic lesions collected from carcasses during postmortem examination. A total of 58 different flocks categorized into pullets, broiler, turkey and duck suspected of MD were sampled using PCR-techniques. Forty three out of the 58 flocks sampled were positive for MDV employing first and nested PCR analysis using serotype-specific primer pairs in the nested PCR, 38 (65.5%), 22 (37.93%) and 20 (34.48%) were positive for MDV-serotypes 1, 2 and 3 respectively. The distribution of three serotypes of MDV was quite different among investigated poultry flocks. Fourteen (73.68%), 7 (36.84%) and 6 (31.57%) farms were positive for MDV-serotypes-1, 2 and 3 respectively in pullet flocks with age range between 8-20 weeks. In layer flocks, 22 (66.66%), 13 (39.39%) and 11 (33.33%) were positive for MDV-serotypes 1, 2 and 3. One broiler flock was positive for MDV-serotype-2 and 3, only one turkey flock positive for MDV-serotypes 1, 2 and 3, one duck farm was positive for MDV- serotype 1. Multiple infections involving two or three of the serotypes were observed in 8-20 weeks old pullets with 4 (21.1%) of the flocks showing MDV-1 and MDV-2, 3 (15.79%) with MDV-1 and 3 and 3 (15.79%) with all the three-serotypes. This study revealed increased virulence of MDV-serotypes-2 and 3 that have been classified as non-oncogenic in field MD-outbreaks.