Abstract
Allopolyploidy is an important force in plant evolution, yet studying natural allopolyploid species remains challenging due to the complexity of their genetic architecture and a lack of reference genomes. A major obstacle is the accurate phasing of subgenomes, which is a prerequisite for the application of diploid‐based population genetic tools. Here, we developed allosplitter , a novel bioinformatic tool that enables precise subgenome phasing for allotetraploids using only genotyping‐by‐sequencing (GBS) data from a derived polyploid and its diploid progenitors. We applied allosplitter to the allotetraploid Achillea wilsoniana and its progenitors ( A. acuminata and A. asiatica ). We uncovered clear asymmetric subgenome evolution: subgenome C (derived from A. acuminata ) exhibited significantly lower genetic diversity, higher population differentiation and higher Tajima's than subgenome Y (derived from A. asiatica ). This indicated a dominant evolutionary role for subgenome C, while historical introgression from A. acuminata further amplified population divergence. Phylogenetic and structure analyses enabled us to reject the biogeographic origin of A. wilsoniana in the Qinling Mountains, instead supporting its origin in the Hengduan Mountains. This study provides a reference‐genome‐free framework for polyploid genomics and offers new insights into the evolution of allopolyploids.