The adaptability of soybean to be grown at a wide range of latitudes is attributed to natural variation in the major genes and quantitative trait loci (QTLs) that control flowering time and maturity. Thus, the identification of genes controlling flowering time and maturity and the understanding of their molecular basis are critical for improving soybean productivity. However, due to the great effect of the major maturity gene E1 on flowering time, it is difficult to detect other small-effect QTLs. In this study, aiming to reduce the effect of the QTL, associated with the E1 gene, on the detection of other QTLs, we divided a population of 96 recombinant inbred lines (RILs) into two sub-populations: one with the E1 allele and another with the e1nl allele. Compared with the results of using all 96 recombinant inbred lines, additional QTLs for flowering time were identified in the sub-populations, two (qFT-B1 and qFT-H) in RILs with the E1 allele and one (qFT-J-2) in the RILs with the e1nl allele, respectively. The three QTLs, qFT-B1, qFT-H and qFT-J-2 were true QTLs and played an important role in the regulation of growth period. Our data provides valuable information for the genetic mapping and gene cloning of traits controlling flowering time and maturity and will help a better understanding of the mechanism of photoperiod-regulated flowering and molecular breeding in soybean.