Background Dermatophytes are a group of closely related keratinophilic fungi that can invade keratinized humans and animals tissues such as skin, hair and nails causing dermatophytosis. They are an important cause of superficial fungal infection. Findings Conventional methods like potassium hydroxide (KOH) microscopy and fungal culture lacks the ability to make an early and specific diagnosis. In this study we have evaluated nested Polymerase chain reaction (PCR) using primers targeting dermatophyte specific sequence of chitin synthase 1 (CHS1) gene and compared with conventional test. A total of 155 patients clinically suspected with dermatophytosis were included in the study. Of which 105 specimens were skin scrapings and 50 were hair. KOH microscopy, fungal culture and first round and nested PCR were done on clinical specimens, and results compared. Nested PCR for dermatophytes was positive in 83.8% specimens, followed by KOH microscopy (70%), first round PCR (50.8) and fungal culture (25.8). Conclusion Results indicate that nested PCR may be considered as gold standard for the diagnosis of dermatophytosis and can aid the clinician in initiating prompt and appropriate antifungal therapy.