BACKGROUND: This study was performed to evaluate etiologies and secular trends in primary amenorrhea in South Korea. METHODS: This retrospective multi-center study analyzed 856 women who were diagnosed with primary amenorrhea between 2000 and 2016. Clinical characteristics were compared according to categories of amenorrhea (hypergonadotropic/hypogonadotropic hypogonadism, eugonadism, disorders of sex development) or specific causes of primary amenorrhea. In addition, we assessed secular trends of etiology and developmental status based on the year of diagnosis. RESULTS: The most frequent etiology was eugonadism (39.8%). Among specific causes, Mullerian agenesis was most common (26.2%), followed by gonadal dysgenesis (22.4%). Women with hypergonadotropic hypogonadism were more likely to have lower height and weight, compared to other categories. In addition, the proportion of cases with iatrogenic or unknown causes increased significantly in hypergonadotropic hypogonadism category, but overall, no significant secular trends were detected according to etiology. The proportion of anovulation including polycystic ovarian syndrome increased with time, but the change did not reach statistical significance. CONCLUSION: The results of this study provide useful clinical insight on the etiology and secular trends of primary amenorrhea. Further large-scale, prospective studies are necessary.