In recent years, the efficiency and accuracy of QTL analysis for identification of useful traits have been increased by high-throughput genotyping. In a previous study, the genome variation of significant DNA polymorphism was observed in early maturing type rice mutant (EMT) by comparing with that of wild type (WT). For detection of major QTL for flowering time, we constructed a linkage map of 36 InDel- and 6 SNP- markers. In the linkage analysis of F2 plants derived from the cross “WT x EMT”, we have detected one potential QTL region on chromosome 6 by M6-3 marker. Also, the Hd1, which contained the target fragment of M6-3 marker, exhibited the relatively high nonsynonymous substitutions in genes located on chromosomal region from M6-2 to M6-4. To evaluate the reliable allele segregation related to expected Mendelian ratio between M6-3 and its flanking markers, M6-3 marker developed in Hd1 gene exhibited the 1:2:1 ratio as clear monogenic segregation in heterozygous F3 plant. Additionally, we further analyzed the different transcript regulations of OsGI and Hd3a gene related to Hd1 involved in photoperiodic flowering pathway. Although the mRNA levels of Hd1 had no difference between WT and EMT, the Hd3a as downstream effector of Hd1 significantly upregulated in EMT, suggesting that Hd1 gene may become nonfunctional.