Cytokinins (CK) plays an important role in plant growth and development, and cytokinin oxidase/dehydrogenase (CKX) gene family regulates the CKs content by degrading them permanently. Identification of the positive alleles in wheat CKX gene family, their association with yield-related traits and the development of functional markers is the first step in using these alleles in a marker-assisted breeding program (MAB). To identify the positive alleles, we sequenced 48 wheat genotypes for TaCKX gene family by Single Molecule Real-Time (SMRT) technology and explored the allelic variations across the full length of the genes. Consequently, we discovered six polymorphic TaCKX genes forming 12 haplotypes. Separate association analysis for each of the polymorphic genes revealed that haplotype TaCKX1BB_2, TaCKX2AA_2, TaCKX5AA_3, TaCKX9AA_2, and TaCKX11AA_3 are significantly associated with high thousand-grain weight (TGW) and short plant height (PH). Functional markers (InDels for TaCKX1BB, TaCKX2AA and TaCKX5AA, and CAPSs for TaCKX9AA and TaCKX11AA) were developed for all the TaCKX polymorphic genes and were validated on Chinese micro-core collection (n=262) and a GWAS population (n=449). Haplotypes TaCKX2AA_2, TaCKX4AA_2, TaCKX5AA_3 and TaCKX9AA_2 were found significantly associated with TGW and PH in both populations. Whereas, TaCKX1BB_2 in GWAS population and TaCKX11_3 in MCC was significantly associated with TGW and PH. In both populations, the mean values of TGW and PH for cumulative positive haplotypes from chromosome 3A, i.e. TaCKX2AA_2, TaCKX4AA_2, and TaCKX5AA_3 were significantly higher as compared to the cumulative negative haplotypes, and the change in mean values was additive in manner. Since the 1940s, positive haplotype frequencies and TGW has gradually increased in Chinese cultivars. Expression profiling of the haplotypes in the seed samples excised at 2, 4, 6 and 8 DAA depicted that the transcript level of positive haplotypes was significantly lower as compared to the negative haplotypes. We conclude that the haplotypes found in six TaCKX genes are significantly associated with TGW and PH, and the functional markers developed in this study can be used in wheat MAS and MAB programs.