This work aimed to compare nested PCR using novel primers targeting the pan-dermatophyte-specific sequence of the chitin synthase 1 gene (CHS1) with KOH microscopy and culture isolation for diagnosis of clinically suspected onychomycosis. This study was conducted during the period from December, 2012 to October 2013. Forty patients attending Outpatient Dermatology and Andrology Clinic in Benha University Hospital. This study was done on forty patients 15 cases were female and the other 25 cases were males with abnormal nails . Their ages ranged from 22 to 77 years. As many as 19 patients were living in rural areas, while 21 patients came from urban areas. Nail scrapings were collected and examined using direct KOH microscopic examination, culture and PCR using double sets of primers. As regard direct microscopy by KOH examination; 33 (82.50%) cases were positive, while 7 (17.5%) were negative. Culture was positive only in 19(47.5%) of nail samples revealing different fungi. Dermatophytes were isolated from 15(37.5%) cases; most of them were T. mentagrophytes. And in 4 cases the only isolated non dermatophytic organism was Aspergillus Niger spp. (10.00%). Nested PCR was positive in 26 (65.00%) nail samples. Conclusion: It is concluded that nested PCR targeting the CHS1 gene may be considered the gold standard for detection of dermatophytes in patients with onychomycosis and can aid the clinician in initiating prompt and appropriate anti fungal therapy. PCR is a very powerful tool for microbiology and clinical mycology. It can detect very small amounts of nucleic acids. This technique may also play an important role in large-scale studies and in the management of problematic cases of onychopathies,