This study was conducted to identify the quantitative trait loci (QTL) associated with spike length and kernel number by analysis of 122 recombinant inbred lines (RILs) derived from a cross between Korean wheat cultivar and Iksan 370, which large kernel number line carried with the longer spike length and higher kernel number/spike than Korean wheat cultivars. Spike length, kernel number/spike and spike density were investigated from replicated field trials in 2013–14 at upland condition in Iksan, Korea. A genetic map was constructed with 92 microsatellite marker loci and a total of nine QTL were identified on six chromosomes for spike length and kernel number. The three QTL, Xwmc 44, Xbarc 205 and Xgwm 292 were detected on chromosomes 1B and 5D, and explained 9.7–16.5% of phenotypic variation for spike length. Among these QTL, Xwmc 44 and Xgwm 292 also explained 8.2 and 10.8% of phenotypic variation for spike density and kernel number/spike, respectively. Xgwm 135, located on chromosomes 1A, was accounted for 10.9% of phenotypic variation for kernel number/spike. Other two QTL for spike density, Xgwm 495 and Xwmc 285, were mapped on chromosome 4B and 4D, respectively. These QTL explained 6.9–7.9% of phenotypic variation for spike density. Three QTL, Xwmc 44, Xgwm 292 and Xgwm 135 for spike length, kernel number/spike and spike density were applied to validate relationship between genotypes of QTL and 29 Korean wheat cultivars grown for four years on upland conditions. Korean wheat cultivars carried aaa and bbb genotypes based on the order of these three QTL, higher and lower kernel number/spike and spike density than other genotypes, although there was not a significant difference in spike length. These QTL could be used in marker assisted selection wheat lines with higher kernel numbers in Korean wheat breeding programmes.