Agropyron cristatum (L.) can be used as a valuable gene resource for broadening the genetic basis of common wheat in breeding programs. Pubing3228 is a new elite wheat-Agropyron hybrid germplasm that possesses many desirable agronomical traits, includingthe grain number per spike (GNS), the main yield component. Uncovering the genetic architecture of GNS in Pubing3228 is important for increasing wheat yield through molecular assisted breeding. In the present study, genetic regions and alleles associated with high GNS from Pubing3228 were analyzed using a recombination inbred line (RIL) population derived from the cross between Pubing3228 and Jing4839. In total, 75 QTLs were identified for GNS. These QTLs mapped to 21 chromosomes (i.e.,all except for 5B and 6A) and explained 3.30–17.17% of the total phenotypic variation. Five QTLs (QGns.wa-1D, QGns.wa-5A, QGns.wa-7D.1, QGns.wa-7D.2, and QGns.wa-7D.3) explained more than 10% of the phenotypic variation in at least two environments. Pubing3228 contributed 94.67% of the total number of QTLs associated with GNS. Candidate gene analysis was conducted for the stable QTLs, and 11 candidate genes were obtained for GNS. Among these 11 genes, a senescence-associated protein gene (TraesCS7D01G148000) linked with the most significant single nucleotide polymorphism (SNP) AX-108748734 on chromosome 7D was relevant to remobilizing the nutrients from the senescing tissues to the developing seeds. The findings of this study will not only provide new insights into the genetic mechanism of high GNS of Pubing3228 but willalso be useful for marker-assisted selection for yield improvement in breeding practice.