The rice cultivar Hikarishinseiki, a semidwarf isogenotype of Koshihikari carrying the Green Revolution sd1 gene, is increasingly grown in both Japan and the United States. Here, we report DNA diagnosis for Hikarishinseiki targeting its Jukkoku-type sd1 locus, which codes for a defective gibberellin 20-oxidase, with a 1 bp substitution in exon 1 (Jukkoku-type GA20ox-2 mutant allele: Jukkoku_GA20ox-2). An allele-specific primer (ASP)-polymerase chain reaction (PCR) with primers SD1F3 and SD1JR gave a PCR product specific to Jukkoku_GA20ox-2. In addition, ASP-PCR with primers SD1F3 and SD1NRM (which contains a mismatch at the third nucleotide from the 3'-terminus of SD1NR) gave a PCR product specific to non-Jukkoku_GA20ox-2. Multiplex ASP-PCR using SD1F3, VIC dye-labeled SD1JR, and FAM dye-labeled SD1NRM enabled simultaneous codominant detection of Jukkoku_GA20ox-2 and non-Jukkoku_GA20ox-2 among 188 cultivars. Also, Hikarishinseiki is identifiable by RM253 polymorphism from 11 cultivars carrying Jukkoku_GA20ox-2. Taken together, our results establish a methodology for distinguishing Hikarishinseiki.