Plant specific protein kinase, SnRK2s, is a major family of signaling genes involved in metabolic regulations, nutrient utilization and response to external stimuli. In the present study, three copies of TaSnRK2.9 were isolated from chromosomes 5A, 5B and 5D of common wheat. The coding regions of TaSnRK2.9-5A, TaSnRK2.9-5B and promoter region of TaSnRK2.9-5D were cloned for sequence polymorphism assay. Gene structure analysis revealed that TaSnRK2.9-5A and -5B contained eight introns and nine exons. Three single nucleotide polymorphisms (SNPs) at 308, 698 and 1700 nt were identified for TaSnRK2.9-5A; however, no polymorphism was identified in TaSnRK2.9-5B and TaSnRK2.9-5D promoter. For high throughput genotyping, two kompetitive allele specific PCR (KASP) markers were developed. Four haplotypes Hap5A-1, Hap-5A-2, Hap-5A-3 and Hap-5A-4 were detected in wheat populations collected from China, Europe and Pakistan. Association assay was performed with mixed linear model in TASSEL (v 5.0), and the results showed that Hap-5A-1/2 of TaSnRK2.9-5A were significantly associated with high thousand kernel weight, while Hap-5A-4 with high grain number per spike. Overexpression of TaSnRK2.9-5A in rice resulted in higher grain number per panicle which is consistent with association assay. Geographic distribution and allelic frequency indicted that the favored haplotypes were positively selected in Chinese (Hap-5A-1/2), Pakistani (Hap-5A-1), east European (Hap-5A-1) and west European (Hap-5A-4) wheat breeding. The results suggest that the developed KASP markers can be utilized in yield improvement by marker assisted selection in wheat breeding.