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AIM: To compare fungal culture and <i>in vivo</i> confocal microscopy(IVCM)in the diagnosis of non-primary fungal keratitis. <p>METHODS: The clinical data of 31 cases(31 eyes)with non-primary fungaI keratitis from September 2016 to February 2017 in our HospitaI were retrospectiveIy reviewed. The positive rate of the two methods was compared by chi-square test. <p>RESULTS: The positive rate by fungal culture was 58%(18/31)and IVCM was 19%(6/31); the positive rate comparison difference was statistically significant between fungal culture and IVCM(<i>χ</i>²=7.56,<i>P</i><0.01). In the 13 eyes with positive culture results, 2 eyes were positive by IVCM; in the 25 positive IVCM eyes, 14 eyes were positive in culture. <p>CONCLUSION: The positive rate of fungal culture in non-primary fungal keratitis is higher than that of IVCM. Fungal culture is an essential auxiliary examination in the diagnosis of non-primary fungal keratitis. With the characteristics of fast, noninvasive and repeatable, IVCM also plays an important role in the diagnosis of non-primary fungal keratitis. The combination of the two methods can improve the positive rate of diagnosis.