Marek's disease (MD) remains a significant concern in commercial poultry, with comb lesions serving as important diagnostic indicators. This study aimed to investigate the prevalence and characterize the histopathological features of MD-associated comb lesions in commercial layer chickens. Fifty comb samples exhibiting gross MD-like lesions were collected from 25 commercial layer farms in Namakkal District and Thalaivasal (Salem District), Tamil Nadu, India. Affected birds ranged from 36 to 58 weeks of age. Samples underwent comprehensive gross pathological examination, histopathological analysis with standardized scoring, and molecular confirmation using polymerase chain reaction (PCR). Gross examination revealed characteristic pale, thickened comb bases in the majority of cases, with some birds showing additional swelling, ulceration, and scab formation. Histopathological examination confirmed MD in 20 of 50 samples (40% positivity rate). A standardized scoring system (0-5) was developed based on pleomorphic lymphoid cell (PLC) infiltration patterns. Distribution of lesion scores was: Score 0 (no lesions) - 30 cases (60%); Score 1 (multifocal mild perivascular PLC infiltration) - 10 cases (20%); Score 2 (focal moderate perivascular PLC infiltration with vasculitis and hyperkeratosis) - 3 cases (6%); Score 3 (multifocal moderate perivascular PLC infiltration) - 2 cases (4%); Score 4 (diffuse moderate perivascular PLC infiltration) - 3 cases (6%); and Score 5 (diffuse severe PLC infiltration extending to comb center with vascular congestion) - 2 cases (4%). Progressive disease severity correlated with increasing lymphoid cell infiltration from perivascular regions to central comb tissue. PCR analysis provided molecular confirmation of MD virus presence in histologically positive cases. This study establishes a 40% diagnostic confirmation rate for clinically suspected MD cases in commercial layers from Tamil Nadu. The developed histopathological scoring system effectively categorizes disease severity based on lymphoid infiltration patterns. Combined histopathological and molecular diagnostic approaches provide robust confirmation of MD, contributing to improved disease surveillance and control strategies in commercial poultry operations. The predominant involvement of mature laying hens (36-58 weeks) suggests the importance of monitoring MD in older commercial flocks.