This study was conducted to collect data from various regions of Jejudo Island, Korea to establish a more accurate and complete academic listing of mushrooms on the island by identifying and organizing them using base sequence analysis. The investigation was performed over five years, from 2019 to 2023, and included a total of nine sites in the southern, western, and eastern areas of Jejudo Island. In total, 481 unique mushroom varieties were collected during the study. These mushrooms included 387 specimens that were classified into 6 classes, 18 orders, 55 families, 118 genera, and 201 species, and 94 unclassified specimens for which the precise species was not known. The collected mushrooms classified by ecotype consisted of 148 species of saprophytes, 47 species of symbionts, and 6 species of parasites. Differences in occurrence patterns were also observed depending on topographical characteristics and vegetation. Finally, three climate-sensitive biological indicator species and two candidate species were also collected, as well as the tropical mushroom Macrocybe gigantea. This study identified 91 species of mushrooms that were previously unreportedon the Jejudo Island. According to the current findings and those previously reported, 7 classes, 26 orders, 123 families, 399 genera, and 1,102 species of mushrooms have been compiled on Jejudo Island to date.