Abstract: Backcrossing was an effective way to breeding high-protein soybean lines. In this study Zhonghuang13 popularized in Yellow-Huai-Hai Valley with super synthetic characters was choose to be the recurrent parent of four crosses. Zhonghuang20 and Dongshan69 which were lower significantly than recurrent parent and Chihuangdou-1 and Taixingniumaohuangyi which were higher significantly than recurrent parent on protein content were screened from soybean mini core collection to be the donor parents. The RP, DP, F2, BC1F2 and BC2F2 of four crosses were conducted to evaluate the genetic variation of protein content and correlation between protein content and main agronomic characters. The results show that the level of recurrent parent and donor parent on protein content would notably affected the performance of hybrid and backcross lines. The mean protein content and the ratio of transgress individuals of hybrid and backcross lines were superior in the cross which recurrent parent and donor parent were both rich in protein. The genetic variation of populations would decrease with backcrossing, in this study variation coefficient of protein content in F2、BC1F2 and BC2F2 descended in turn. The mean protein content and variation coefficient of BC2F2 were both closed to recurrent parent. The protein content of F2 populations obedient to normal distribution, the BC1F2 populations of two crosses which donor parents were significantly higher than recurrent parent obedient to skewed distribution, but it recovered normal distribution in BC2F2. F2 was positively correlated with donor parent significantly on main agronomic characters such as protein content, oil content, plant height, height of 1st pod , No. of stem node , effective branch number, pod No. per plant, seed No. per plant, 100 seed weight. BC1F2 and BC2F2 were positively correlated with donor parents significantly on protein content, oil content, plant height, pod No. per plant, seed No. per plant, 100 seed weight.