Soybean agglutinin (SBA), a major anti-nutritional factor in soybeans, seriously affects the safety of and effects on feed animals. Soybean varieties with lower SBA contents could play a positive role in the promotion of the fodder industry. In the present study, one male parent, Hefeng 45, was used in three recombinant I bred line (RIL) populations to detect quantitative trait loci (QTL) associated with SBA content across three different environments. As a result, a total of six, six, and four QTL related to SBA content were identified in the groups Hefeng 45 × Zhongdou 27 (HZ), Hefeng 45 × Dongnong 48 (HD), and Hefeng 45 × Taipingchuan Blacksoy (HT), respectively. Most of the QTL underlying SBA explained less than 10% of the phenotypic variation, but some major QTL with higher additive effects have stable expressions across different environments and RIL populations. On the contrary, many QTL dependent on the environment or RIL populations showed mainly weak effects, and gene × environment interaction occurred in the opposite direction to additive effects and (or) epistasis effects. Two (SbaHZC1-1 and SbaHZD1b-1, linked to Satt139 and Satt189, respectively) and one (SbaHDD1a-1, linked to Satt402) QTL for SBA were identified in HZ and HD populations, respectively, across three environments. Those QTL (Satt139 and Satt189) might have potential in the application of marker-assisted selection for low agglutinin content soybean.