The Shitake mushroom (Lentinula edodes) is the second most-consumed mushroom in the world; in Colombia, it is cultivated and commercialized on a small scale in some supermarkets. Little is known about the precedence, nutritional and medicinal properties of Shiitake produced in Colombia. In this study, four shiitake isolates were grown in Colombia (LEUCO1, LEUCO2, LEUCO3, and LEUCO4) were sequenced in their ITS genes and evaluated for the production of three medicinal metabolites, eritadenine, ergotioneine and β-glucans (1,3-1, 6), using submerged culture. Genetic analysis revealed that all the isolates were close and related to the Japanese strain Cr62. LEUCO1 and LEUCO2 showed a distance of 0.000, as well as LEUCO3 and LEUCO4. All four isolates produced erythadenin in a range of 26.3-8.6 mg / L, with the best performance of LEUCO1 at 26.3 mg / L (p <0.05). Ergotioneine was produced with similar statistical yields in all the isolates with an average of 0.7 mg / g of dry weight biomass (DW). Β-glucans (1.3-1.6) were produced with yields of 5.6 - 3.8% of DW biomass, with the best values ​​for LEUCO2 and the lowest for LEUCO4 (p <0.05). In conclusion, we identified low genetic diversity in the four isolates, corresponding to two haplotypes with minimal genetic difference between them, related to the Japanese strain Cr62, indicating that Colombian farmers cultivate almost the same strains of shitake. Secondary metabolites, eritadenine, β-glucans and ergotioneine were found in promising yields useful for the pharmaceutical and food industries. More studies should be conducted to improve the yield of shitake metabolites through new growing conditions for industrial production and to find metabolic pathways and related genes.