Nucleolar dominance (ND) is a widespread epigenetic phenomenon in hybridizations where nucleolus transcription fails at the nucleolus organizing region (NOR). However, the dynamics of NORs during the formation of Triticum zhukovskyi (GGA^uA^uA^mA^m), another evolutionary branch of allohexaploid wheat, remains poorly understood. Here, we elucidated genetic and epigenetic changes occurring at the NOR loci within the A^m, G, and D subgenomes during allopolyploidization by synthesizing hexaploid wheat GGA^uA^uA^mA^m and GGA^uA^uDD. In T. zhukovskyi, A^u genome NORs from T. timopheevii (GGA^uA^u) were lost, while the second incoming NORs from T. monococcum (A^m A^m) were retained. Analysis of the synthesized T. zhukovskyi revealed that rRNA genes from the A^m genome were silenced in F₁ hybrids (GA^uA^m) and remained inactive after genome doubling and subsequent self-pollinations. We observed increased DNA methylation accompanying the inactivation of NORs in the A^m genome and found that silencing of NORs in the S₁ generation could be reversed by the cytidine methylase inhibitor. Our findings provide insights into the ND process during the evolutionary period of T. zhukovskyi and highlight inactive rDNA units may serve as a "first reserve" in the form of R-loops, contributing to the successful evolution of T. zhukovskyi.