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Purpose: To highlight the usefulness of polymerase chain reaction (PCR) for the rapid diagnosis of systemic fungal infections. Methods: Clinical samples were collected from 50 clinically suspected cases of systemic mycosis and subjected to smear, culture, antifungal sensitivity and PCR (based on 18S rRNA gene). Results: Of the 50 clinical specimens tested by PCR, 39 were found to be positive. PCR gave more positive results than smear and culture examination. Out of the 50 clinical specimens 35 were found to be fungal culture positive. The sensitivity testing results of these fungal isolates showed that there was a good correlation between the in vitroresults and the clinical response of the patient to antifungal therapy. Itraconazole exhibited maximum antifungal activity followed by fluconazole, ketoconazole and amphotericin B. Conclusions: PCR technology provides rapid and accurate diagnosis of fungal infection, however, it must be used with caution to avoid false positives.