Purpose. To assess the utility of polymerase chain reaction (PCR) in diagnosing fungal keratitis and compare its sensitivity and specificity with those of the conventional microbiologic techniques used in the authors’ laboratory. methods. A prospective nonrandomized investigation was undertaken at a tertiary-care ophthalmic facility to evaluate 40 eyes of 40 patients with presumed fungal keratitis, both fresh and treated. Besides routine bacterial culture and sensitivity, corneal scrapings were evaluated by fungal culture, potassium hydroxide (KOH) wet mount, Gram’s stain, and PCR. The conventional PCR technique was followed with minor modifications to suit the setup, using primers targeted to 28S rRNA sequence, which is common to all fungi that cause corneal infections in tropical climes. results. Of the 40 presumed cases of mycotic keratitis (30 untreated), PCR showed positivity in 50%, culture in 25%, hyphae in KOH in 40%, and Gram’s staining in 35%. The sensitivities of PCR, KOH, and Gram’s were 70%, 60%, and 40% and specificities 56.7%, 66.7%, and 66.7%, respectively. Among 10 of 40 eyes pretreated with antifungal agents, PCR was positive in 50%, but culture in 30%. The time taken for PCR assay was 4 to 8 hours, whereas positive fungal cultures took at least 5 to 7 days. conclusions. PCR not only proved an effective rapid method for the diagnosis of fungal keratitis, but was also more sensitive in our hands than KOH wetmount and Gram’s smear. Barring the potential limitations, PCR remains a promising tool for faster diagnoses of fungal keratitis.