Hypoxia caused by water seeding of rice inhibits germination and often leads to serious problems in seedling establishment and early growth. To solve this problem, it is necessary to develop rice cultivars that can stably germinate and grow under anaerobic environments. In this study, we performed QTL analysis on anaerobic germination (AG) tolerance using 139 recombinant inbred lines (RILs) derived from a cross between Milyang23 and Gihobyeo, and identified two QTLs (qAG2.1, qAG2.2) on chromosome 2. The LOD scores at qAG2.1 and qAG2.2 were 3.30 and 5.31, respectively. The phenotypic variances explained by the QTLs were 9.19% and 14.99%, respectively. The japonica cultivar Gihobyeo provided alleles for AG tolerance at both QTLs. While the chromosomal location of qAG2.2 overlapped with previously identified QTLs for AG tolerance, qAG2.1 was detected in a locus that has not been reported previously. The RILs carrying favorable type alleles for the AG tolerance at both QTLs (qAG2.1 + qAG2.2) expressed 41.0% survival rate under the AG condition, which was significantly higher than those of the RILs carrying single QTL, qAG2.1 (22.0%) or qAG2.2 (26.2%). As the favorable alleles for the two QTLs found in this study derived from the japonica cultivar Gihobyeo, it is expected that they would accompany less linkage drag such as shattering and lodging compared to those derived from indica or landraces. The result of this study would provide useful information for improving AG tolerance of japonica rice cultivars that can be used for water seeding cultivation.