In order to screen the aroma characteristics of soybean, a new method was established which can quickly quantify the content of 2-acetyl-1pyrroline (2-AP), an important compound related to soybean aroma, using gas chromatography-mass spectrometry (GC-MS). Based on peak profile, total peak area and retention time as test indexes, an accurate identification method for fragrant soybeans was established. The optimum parameters of the protocol consisted of column temperature 70°C, sample injector temperature 180°C, optimum extraction alcohol content 1 mL, NaCl content 0.1 g, ultrasonication time 10 min, and extraction time 1 h, which were established by using the orthogonal test of single factors and three factors with four levels (L9(3)4). 2-AP content of leaves had significant correlations with seeds, which were easier to measure. The protocol was simple and easy to carry out, consumed only small amounts of reagents, and provided accurate and reliable results with good reproducibility. A total of 101 soybean genotypes from different geographical sources were analyzed using this protocol. The results showed that the average content of 2-AP was 0.29 mg L–1, ranging from 0.094 to 1.816 mg L–1, and the genetic diversity index was 0.54. Among all genotypes-tested, they were classified into three grades, including seven elite genotypes identified as “grade one fragrant soybeans”, which were Zhonglong 608, Heinong 88, Ha13-2958, Hongmiandou, Heinong 82, Huangmaodou, and Jiyu 21. These results provide both an identification technique and several elite aroma genotypes for gene discovery and good quality breeding in soybean.
