Branch number (BN) is an important agronomic attribute related to the plant architecture, adaptability, and yield of soybean. To date, few studies of BN have been conducted to elucidate its genetic background. We aimed to localize genetic factors affecting BN using segregating populations derived from the high-branching cultivar'Kennong24' (KN24) and the low-branching cultivar 'Kenfeng19' (KF19). Composite interval mapping analysis detected a QTL (qBN-1) on chromosome 6 between the SSR markers BARCSOYSSR_06_0993 and BARCSOYSSR_06_1070 using an F2 population. To fine-map qBN-1, a RIL population was developed and genotyped with 14 SSR markers located in the QTL region. qBN-1 was localized to a 115.67-kb interval flanked by markers BARCSOYSSR_06_1048 and BARCSOYSSR_06_1053. The QTL was further confirmed using backcross populations of size 1305 (BC2F2 with KN24 as a recurrent parent) and 1712 (BC3F2 with KF19 as a recurrent parent). The fine-mapping region of qBN-1 contained only two candidate genes, Glyma.06G208800 and Glyma.06G208900, whose expression patterns were investigated by qRT-PCR. Compared to Glyma.06G208800 gene expression, Glyma.06G208900 showed the highest expression of the two genes and showed a significant difference in expression between high-and low-branching genotypes in either axillary meristem or shoot apical meristem, and showed opposite expression patterns in the two tissues at V4 and R1 stages. These results identify Glyma.06G208900 as a novel candidate gene controlling BN. Taken together, the results of this study provide a foundation for cloning and functional analysis of the qBN-1 gene and for the improvement of BN by marker-assisted selection in soybean breeding.