Flooding stress causes a significant reduction in soybean yield. The development of flood-tolerant cultivars is an effective way to minimize yield loss due to flooding. Information on candidate genes for flooding tolerance is useful for developing tolerant lines. The objective of this study was to identify potential candidate genes for flooding tolerance in soybean by integrating the results of a quantitative trait locus analysis and RNA sequencing. A total of 19 genes showed good amplification in capillary electrophoresis and were further analyzed through a reverse transcription quantitative polymerase chain reaction (qRT-PCR); two of these genes showed differential expression among tolerant and susceptible lines. The expression of Glyma.12g030900 and Glyma.10g050300 in leaf and root tissues, respectively, was higher in several tolerant lines than in the susceptible lines under flooding stress. The chlorophyll index of the tolerant lines was also consistently higher than that of the susceptible lines over two years, supporting the qRT-PCR results. This study provides useful information on flooding tolerance in soybeans.