In order to study the genetic stability of 5Ag chromosome from Thinopyrum ponticum in different wheat backgrounds and its transmission through gametes， the wheat-Th.ponticum disomic substitution line DS5Ag（5D） （Bainong Puyan 5814， PY5814） was hybridized with six wheat cultivars（lines）： Chinese Spring， Bainong Aikang 58， Lankaoaizao 8， Wenmai 6， Zhoumai 16， and Q03073. The F₁ hybrids were selfed and reciprocally crossed with the six aforementioned varieties（lines）， the F₂ and BC₁F₁ generations were genotyped using molecular marker technology. Results showed that all F₁ hybrids derived from PY5814 and the six wheat varieties（lines） exhibited normal seed set. In the F₂ segregation populations of different crosses， the transmission rate of the 5Ag chromosome ranged from 51.61% to 71.76%， indicating that the transmission rate of 5Ag chromosome was different in different wheat genetic backgrounds， with its transmission being influenced by the wheat genetic background. In BC₁F₁ populations， the transmission rates of the 5Ag chromosome through female and male gametes ranged from 21.43% to 31.73% and 29.51% to 39.73%， respectively， confirming its transmission via both gamete types. Notably， under the genetic backgrounds of Lankaoaizao 8 and Wenmai 6， the male gamete transmission rate exceeded the female gamete rate， suggesting the preferential suitability of this substitution line as a paternal parent in genetic studies. At the same time， some F₂ individuals were verified by genomic in situ hybridization （GISH）， and the results were consistent with the results of molecular markers. Furthermore， this study also offers theoretical guidance for predicting and establishing an appropriate hybrid population size， thereby enabling the selection of target progenies carrying the 5Ag chromosome.