A total of 223 accessions of core collection using a glyphosate tolerance transgenic line AG5601 as a control, were used for comparison of the glyphosate tolerance. They were treated with glyphosate (roundup) on cotyledonary node as the target point in the early stage of germination of soybean using four different concentrations of roundup dilute solution (1/10, 1/100, 1/1000, 1/10000). The results showed that highest concentration (1/10 roundup dilution) resulted soybean plant height significantly reduced but both 1/1000 and 1/10000 roundup dilution had no significant influence on plant growth, indicating the glyphosate tolerance of soybean would decrease with the concentration of glyphosate increased. However, there were variations of tolerance among different soybean accessions when they treated with 1/100 roundup dilution. Then we identified 10 elite accessions with moderate glyphosate tolerance. Despite the glyphosate tolerance of soybean mini core collection was much lower than that of transgenic AG5601, we found out that there were great variations of glyphosate tolerance among different accessions. This study provides information for selection of transformation receptors and parents using transgenic and traditional techniques in development of glyphosate resistant soybeans.