In this study, we analyzed the growth characteristics and isoflavone content of 43 soybean varieties highly adaptable to highland areas. The flowering period of each cultivation zone was from July 15 to August 12 at Daewallyeong, from July 18 to August 11 at Jinbu, and from July 23 to August 13 at Gangneung. The accumulated temperature from flowering to maturity was 1,297 °C for Daegwallyeong, 1,391 °C for Jinbu, and 1,685 °C for Gangneung. Forty-three varieties were classified into four utilities; soy sauce and tofu, bean sprouts, cooking with rice, and vegetable and early maturity. The content of isoflavone was highest at 2,579 µg/g in varieties for soy sauce and tofu usage. Five varieties (“Paldalkong,” “Sinpaldal2,” “Ilmikong,” “Sinpaldalkong,” and “Daepung”) cultivated in Daegwallyeong had over 4,000 µg/g of isoflavone. The isoflavone content of the region Daegwallyeong was different at the significance level of 0.1 (p=0.061) compared to Gangneung. There was no significant difference between Gangneung and Jinbu. It is thought that the low temperature of the maturation stage during the growing period affected isoflavone accumulation. The varieties with more than 3,000 µg/g of isoflavone content in Daegwallyeong, Jindu, and Gangneung were “L29,” “Williams82,” “Ilmikong,” and “Daepung.” These were genetically and environmentally stable in isoflavone content. It is expected that this study will be used as basic data for the functional breeding and selection of soybean varieties highly adaptable to a specific region, and to help expand soybean cultivation areas in highlands.