Onychomycosis, a fungal nail infection, has become one of the most important dermatophytoses. The current laboratory methods for diagnosis are the potassium hydroxide (KOH) scraping technique and fungal culture. Unfortunately, a predictably successful diagnostic approach to onychomycosis does not yet exist. The purpose of this study was to develop a deoxyribonucleic acid (DNA)-based diagnostic method to improve the sensitivity and specificity of the detection and differentiation of the pathogenic fungi of onychomycosis. This study attempted to detect fungi in the nail using Polymerase Chain Reaction (PCR) primer systems that were designed in conserved sequences of the small ribosomal subunit JBS-rRNA genes shared by mos/ fungi, and differentiated between species by DNA sequencing technique of the amplified product. Material from 50 nails suspected as having onychomycosis and 10 healthy nails as a control were subjected in parallel to the three mycological methods of detection; microscopic examination, culture, and PCR. Fragments of the gene coding for JBS-rRNA were amplified successfully from the suspected nails and DNA sequencing technique was sufficiently successful to allow the recognition of individual species. The combined PCR-DNA sequencing technique appears to be a more sensitive detection and identification technique for onychomycosis than conventional methods and has considerable diagnostic value.