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To investigate the role of in vivo confocal microscopy for detection of Pythium insidiosum keratitis.Medical records and confocal microscopy findings of 20 patients (21 eyes) with culture- or polymerase chain reaction–proven P. insidiosum keratitis diagnosed at the Khon Kaen University Eye Center from January 2009 to December 2015 were retrospectively reviewed. Confocal microscopy was performed using Nidek ConfoScan 4. The images from 21 eyes with P. insidiosum keratitis and 4 other fungal keratitides were analyzed visually for morphology of the hyphae and special characteristics that would enable physicians to distinguish P. insidiosum keratitis from other fungal keratitides.A total of 21 eyes of 20 patients were included in the study. Fourteen (70%) were men, and 6 (30%) were women. In vivo confocal microscopy was able to identify hyphae in 20 of 21 eyes (95%). Beaded string-like hyperreflective branching structures with mean branching angles at 78.6 degrees or thin hyperreflective long lines were found in confocal microscopy findings of P. insidiosum keratitis. The diameter of the hyphae varied from 1.5 to 7.5 μm.P.insidiosum keratitis is a severe progressive infectious corneal disease that causes vision loss in most patients. Confocal microscopy may provide rapid in vivo visualization of P. insidiosum hyphae in corneal tissues, but it cannot distinguish P. insidiosum from other fungal keratitides. Culture identification with zoospore induction or polymerase chain reaction remains the most reliable means to confirm the diagnosis of P. insidiosum keratitis.