Ratooning ability is a key factor that influences ratoon rice yield, in the area where light and temperature are not enough for second season rice. In the present study, an introgression line population derived from Minghui 63 as the recipient parent and 02428 as the donor parent was developed, and a high-density bin map containing 4568 bins was constructed. Nine ratooning-ability-related traits were measured, including maximum tiller number, panicle number, and grain yield per plant in the first season and ratoon season, as well as three secondary traits, maximum tiller number ratio, panicle number ratio, and grain yield ratio. A total of 22 main-effect QTLs were identified and explained for 3.26–18.63% of the phenotypic variations in the introgression line population. Three genomic regions, including 14.12–14.65 Mb on chromosome 5, 4.64–5.76 Mb on chromosome 8, and 10.64–15.52 Mb on chromosome 11, were identified to simultaneously control different ratooning-ability-related traits. Among them, qRA5 in the region of 14.12–14.65 Mb on chromosome 5 was validated for its pleiotropic effects on maximum tiller number and panicle number in the first season, as well as its maximum tiller number ratio, panicle number ratio, and grain yield ratio. Moreover, qRA5 was independent of genetic background and delimited into a 311.16 kb region by a substitution mapping approach. These results will help us better understand the genetic basis of rice ratooning ability and provide a valuable gene resource for breeding high-yield ratoon rice varieties.