Root systems that improve resource uptake and penetrate compacted soil (hardpan) are important for improving soybean (Glycine max L. Merr.) productivity in optimal and sub-optimal environments. The objectives of this research were to evaluate a soybean germplasm collection of 49 genotypes for root traits, determine whether root traits are related with plant height, shoot dry weight, chlorophyll index, and seed size, and identify genotypes that can penetrate a hardpan. Plants were maintained under optimal growth conditions in a greenhouse. Single plants were grown in mesocosms, constructed of two stacked columns (top and bottom columns had 25 and 46 cm height, respectively, and 15 cm inside diameter) with a 2-cm thick wax layer (synthetic hardpan; penetration resistance, 1.5 MPa at 30°C) in between. Plants were harvested at 42 days after planting. Significant genetic variability was observed for root traits in the soybean germplasm collection, and genotypes that penetrated the synthetic hardpan were identified. Genotypes NTCPR94-5157, NMS4-1-83, and N09-13128 were ranked high and PI 424007 and R01-581F were ranked low for most root traits. Shoot dry weight and chlorophyll index were positively related with total root length, surface area, and volume, and fine root length (Correlation coefficient, r ≥ 0.60 and P-value < 0.0001 for shoot dry weight and r ≥ 0.37 and P-value < 0.01 for chlorophyll index]. Plant height was negatively correlated with total root surface area, total root volume, and average root diameter (|r| ≥ 0.29, P-value < 0.05). Seed size was not correlated with any root traits. The genetic variability identified in this research for root traits and penetration are critical for soybean improvement programs in choosing genotypes with improved root characteristics to increase yield in stressful or optimum environments.