The phosphate transporter 1 (PHT1) family are integral membrane proteins that operate in phosphate uptake, distribution and remobilization within plants. In this study, we report on the functional characterization and expression of two PHT1 family members from ryegrass plants (Lolium perenne L.) and determine their roles in the specificity of Pi transport. The expression level of LpPHT1;4 was strongly influenced by phosphorus (P) status, being higher under P-starvation condition. In contrast, the expression level of LpPHT1;1 was not correlated with P supply. Yeast mutant complementation assays showed that LpPHT1;4 can complement the growth defect of the yeast mutant Delta pho84 under Pi-deficient conditions, whereas the yeast mutant expressing LpPHT1;1 was not able to restore growth. Phylogenetic and molecular analyses indicated high sequence similarity to previously identified PHT1s from other species in the Poaceae. These results suggest that LpPHT1;1 may function as a low-affinity Pi transporter, whereas LpPHT1;4 could acts as a high-affinity Pi transporter to maintain Pi homeostasis under stress conditions in ryegrass plants. This study will form the basis for the long-term goal of improving the phosphate use efficiency of ryegrass plants.