BACKGROUND: The accumulation of cadmium (Cd) in ryegrass (Lolium multiflorum Lamk.) as a widely used pasture plant poses a serious risk to food safety. This study aimed to investigate the differences in phenotypes, physiology, and expression of metal transporters between four ryegrass genotypes (diploid/tetraploid and Cd-tolerant/sensitive). RESULTS: The diploid/Cd-sensitive genotypes were found to uptake, accumulate, and translocate more Cd compared to the tetraploid/Cd-tolerant genotypes. Cd with more soluble components facilitated the transfer of Cd from root to shoot in the sensitive genotypes. Tetraploid and Cd-tolerant Chuansi No.1 accumulated less Cd in shoots but higher ratio in root cell wall, making it a promising model for studying the mechanisms of plant resistance to Cd stress. The complex regulatory system and dilution effect contributed to the lower uptake and accumulation of Cd in tetraploid genotypes. Moreover, tetraploid genotypes exhibited higher expression of genes that promoted Cd efflux, which could contribute to their lower Cd accumulation. CONCLUSIONS: Overall, this study sheds light on the physiological and transcriptional mechanisms of Cd uptake and accumulation by different polyploids, providing guidance for ryegrass breeding and soil improvement.