A case of fungal keratitis is presented in which corneal scrapings were obtained for microbiological studies, including morphological identification and molecular characterization of the etiologic agent. Comparative sequence analyses of the Internal Transcribed Spacer domain of 5.8S and 26S regions of nuclear rDNA showed 100% identity with different species of Alternaria and PCR-RFLP analysis of Intergenic Spacer regions revealed intraspecific variation. The combination of morphological and molecular characters resulted in the unambiguous identification of the causal agent as Alternaria alternata. Treatment with antifungals contributed to the improvement in the patient's lesions.