Rapid detection of allelic variation and identification of advantage haplotypes responsible for spike related traits play a crucial role in wheat yield improvement. The released genome sequence of hexaploid wheat (Chinese Spring) provides an extraordinary opportunity for rapid detection of natural variation and promotes breeding application. Here, selection signals detection and genome-wide association study (GWAS) were conducted for spike related traits. Based on the genotyping results by 90K SNP chip, 192 common wheat samples from southwest China were analyzed. One hundred and forty-six selective windows and one hundred and eighty-four significant SNPs (51 for spike length, 28 for kernels per spike, 39 for spikelet number, 30 for thousand kernel weight, and 36 for spike number per plant) were detected. Furthermore, tightly linkage and environmental stability window clusters and SNP clusters were also obtained. As a result, four SNP clusters associated with spike length were detected on chromosome 2A, 2B, 2D, and 6A. Two SNP clusters correlated to kernels per spike were detected on 2A and 2B. One pleiotropy SNP cluster correlated to spikelet number and kernels per spike was detected on 7B. According to the genome sequence, these SNP clusters and their overlapped/flanking QTLs which have been reported previously were integrated to a physical map. The candidate genes responsible for spike length, kernels per spike and spikelet number were predicted. Based on the genotypes of cultivars in south China, two advantage haplotypes associated with spike length and one advantage haplotype associated with kernels per spike/spikelet number were detected which have not been effectively transited into cultivars. According to these haplotypes, KASP markers were developed and diagnosed across landraces and cultivars which were selected from south and north China. Consequently, KASP assay, consistent with the GWAS results, provides reliable haplotypes for MAS in wheat yield improvement.