The influence of the semidwarfing gene sd1 derived from the rice cultivar Jukkoku (Jukkoku sd1) and IR8 (IR8 sd1), which contributed to the Green Revolution, d60 from Hokuriku 100, as well as the combination of sd1 and d60 (Jukkoku sd1 plus d60 and IR8 sd1 plus d60), was investigated using isogenic lines raised by backcrossing with the cultivar Koshihikari. The isogenic lines carrying Jukkoku sd1, IR8 sd1, d60, Jukkoku sd1 plus d60, and IR8 sd1 plus d60 had considerably shorter culm lengths than Koshihikari by 19.2%, 22.8%, 26.0%, 45.1%, and 43.4%, respectively. The sd1 plus d60 lines showed additively reduced culms, indicating that the function of d60 was different from sd1. In contrast to the culm reduction, Jukkoku sd1 showed productive merit with a panicle length of 2.5% greater than the origin. MiSeq next-generation sequencer was used to optimize a minimum scale to detect Jukkoku sd1 in practical breeding. Mapping with the reference genome of Nipponbare gained the average depths of Koshihikari Jukkoku sd1 and Koshihikari being 9.17 and 7.29, respectively. Comparing the vcf files of the entire genomes of Koshihikari Jukkoku sd1 and the virtual Koshihikari revealed a G to T SNP at position 38,382,746 in the sd1 locus on chromosome 1 of Koshihikari, causing a loss-of-function mutation of GA20-oxidase.