In this study, a total of 87 markers (11 SSRs and 76 SNPs) associated with flowering time of soybean were identified via GWAS. The number and effect of loci associated with flowering time of soybean depended on the photo-thermal conditions. The loci with large effects were found to be located on Gm 11, Gm 16 and Gm 20, consistent with previous reports. The variation of soybean flowering time among the cultivars mainly resulted from gene × environment interactions, particularly epistasis × environment interaction and additive × environment interaction. Gm04_4497001 (close to GmCOL3a), Gm16_307609 (close to GmFT2a and GmFT2b), and Gm19_47514601 (close to E3 or GmPhyA3) are important for controlling flowering time. Among them, Gm04_4497001 may be the major locus with epistatic interaction with other loci for controlling flowering time. The direction and magnitude of the interaction between loci and environments were dependent on photo-thermal conditions, indicating that photoperiod determines the developmental status of plant (vegetative or vegetative), but temperature controls the developmental rate of plant. In summary, the results provide insights into the genetic basis of soybean flowering time and markers could be used for marker-assisted breeding to improve soybean adaptation.