SUMMARYNucleolar dominance (ND) is a widespread epigenetic phenomenon in hybridizations where nucleolus transcription fails at the nucleolus organizer 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 a cytidine methylase inhibitor. Our findings provide insights into the ND process during the evolutionary period of T. zhukovskyi and highlight that inactive rDNA units may serve as a ‘first reserve’ in the form of R‐loops, contributing to the successful evolution of T. zhukovskyi.