Increasing grain yield potential and stability is deemed as a pivotal goal in modern durum wheat plant breeding since an increase of at least 60% in wheat production is strongly required to meet an expected rise in demand resulting from the massive global human population growth by 2050. Numerous studies demonstrated that grain yield is a complex multifactorial trait, resulting from the interaction and correlation among three major components, namely kernel number per spike (KNS), kernel weight (KW), and the number of spikes per unit area (SNM). For purposes of genetic improvement of grain yield, the direct selection of yield is not always effective. Therefore, the most desirable approach to improve grain yield is through selection for grain yield-related traits. The dissection of grain yield into grain yield components also facilitates the investigation of the genetic control of such traits, up to the identification of the relevant loci and the candidate and causal genes underlying the single yield components. The initial explorative mapping study was conducted based on 338 RILs derived from a four-way cross among diverse elite highly differentiated grain yield components. Parents of the RIL population were cultivars Neodur (French cv), Claudio (Italian cv), Colosseo (Italian cv), and Rascon/2*Tarro (CIMMYT cv) (NCCR Population). The RIL was characterized for a range of agronomic traits over two consecutive years in two locations of the Po Valley, Italy. Out of the association analysis, a highly significant SNP-based haplotype underlying the number of kernels per central spikelet (KNSL) and the number of kernels per spike (KNS) was detected on the centromeric region of chromosome 2A, named GNI2. A favorable allele (+0.56 grain per central spikelet) was identified in the CIMMYT parent Rascon/2*Tarro. As a follow-up, in this study, the objective was to obtain heterogeneous inbred families (HIF) that segregated for the SNPs-based haplotype by designing 2 KASP markers converted from two 90K-SNPs markers to narrow down the investigated region of interest to 4 Mb. A population of 1,500 F4 lines, developed from a cross between two elite durum wheat cultivars with a contrasting SNPs-based haplotype in the target region (Relief and Iride), was genotyped with the designed KASP markers, thereby identifying several HIF. Subsequently, marker-assisted selection (MAS) led to obtaining three consolidated pairs of homozygous HIF-derived near-isogenic lines (HIF-NILs) that were seed increased and validated for increased fertile florets and grain number per spikelet. These selected lines will be employed for RNA-seq assays to obtain a transcriptome profiling of the gene network controlled by the investigated genomic region. Additionally, the HIF-NILs will be used to conduct physiological studies on the effects of the increased sink demand driven by the Rascon/2*Tarro plus allele.