Genetic improvement of root traits in soybean largely relied on exploring the elite loci that associated with beneficial root traits. In this study, 242 northern spring cultivars and 102 landraces were subjected for analyzing phenotypic variation in root under laboratory conditions in 2018 and 2019. 175 pairs of SSR primers were applied for genotyping, followed by the genetic linkage disequilibrium analysis, the population structure analysis, and association analysis using general linear model (GLM). The results show that both collinear combination and non-collinear combination groups have different degree of linkage disequilibrium. The cultivars were clustered into two subpopulations. Seventeen genetic loci associated with six root traits were detected showing the phenotypic variation ranged from 1.35% to 7.83%. The landraces were assigned into two subgroups. Twelve genetic loci were found to be associated with five root traits, which contributed to 3.89% to 16.64% on phenotypic variation. Two genetic loci Satt309 and Sat_358, which were simultaneously detected in the two cultivar and landrace populations, were associated with main root length and root average diameter, respectively. A total of 38 alleles with positive effect were present in 29 cultivars. Out of them, the alleles of main root length (MRL), root length (RL), root average diameter (RAD), root volume (RV), root surface area (RSA), root tip number (RTN) with the greatest synergistic effect were Satt309-122bp (Hefeng35), Sat_272-254bp (Heihe16), Sat_358-236bp (Jilin12), Sat_381-193bp (Suinong26), Sat_381-193bp (Kendou18), and Sat_381-224bp (Hefeng51), respectively. A total of 33 alleles with synergistic effect were found from 31 landraces. For example, the alleles governing MRL, RL, RAD, RV, RSA, RTN which had the greatest synergistic effects, were Satt220-242bp (Xiaobaiqi), Satt683-206bp (Wangqingshenxiandong), Sat_160-174bp (Pingdingxiang-2), Satt239-200bp (Linjiangdahuangdou), Satt247-245bp (Bailudou-1), and Satt156-227bp (Xiaobaiqi), respectively. Collectively, this study identified the superior alleles and donor lines which could be used in breeding for soybean varieties with superior root traits.