Abstract Background The distribution, accumulation, and toxicological effects of two perennial ryegrass (Lolium perenne L.) varieties under combined cadmium (Cd)-arsenic (As) stress are worth exploring. Two varieties, ʻNicaraguaʼ (high-Cd/As-accumulating, DPB) and ʻVenusʼ (low-Cd/As-accumulating, WNS), were selected as experimental materials for pot trials. Subcellular fractionation, ultrastructural changes, and key transporter proteins cation exchanger (CAX), heavy metal ATPase (HMA), natural resistance-associated macrophage protein (NRAMP), and phosphate transporter (PHT) were analyzed under combined Cd-As stress. Results (1) The translocation factors of perennial ryegrass for Cd and As were