Background: Myiasis is the infestation of flies' larvae in living or dead tissues of the human body and animals. Ophthalmomyiasis is divided into internal and external types and thelarvae penetrate eyes in the internal type. This study aimed to examine larval morphology and molecular identification of flies causing ophthalmomyiasis and determine its incidence rate in referred patients to an ophthalmology clinic in Shiraz during 2019. Materials and Methods: During one year, all larvae were isolated from patients’ eyes. These larvae were identified using the morphological method according to the 1965 diagnostic key of Zumpt. Molecular confirmation was performed using a pair of specific primers for the Cytochrome Oxidase I (COI) gene in the next step. The expected amplicons were sequenced, and their results were aligned and analyzed using the nucleotide BLAST. Results: Overall, 224 fly larvae were isolated from 36 patients. According to the morphological analysis, all larvae were Oestrus ovis. Molecular analysis confirmed morphological results. Patients presenting acute conjunctivitis symptoms had a mean age of 34 ± 2 years, and there were four women (11%) and 32 men (89%). The highest incidence were recorded in the autumn season (55.8%). Morphological results of flies were confirmed by amplifying the expected size of the CO1 gene through conventional PCR. Conclusion: The cases of ophthalmomyiasis are higher than those mentioned in the published articles and this might be due to the absence of a regular monitoring program to care for the ophthalmomyiasis cases in the public health system. Therefore, due to the importance and potential incidence of this disease in Fars province, establishing a disease care program is necessary for the health surveillance system.