Genetic variation in phosphorus (P) efficiency exists among wheat (Triticum aestivum) and barley (Hordeum vulgare) genotypes, but the underlying mechanisms for the variation remain elusive. High- and low-affinity phosphate (Pi) PHT1 transporters play an indispensable role in P acquisition and remobilization. However, little is known about genetic variation in PHT1 gene expression and association with P acquisition efficiency (PAE) and P utilization efficiency (PUE). Here, we present quantitative analyses of transcript levels of high- and low-affinity PHT1 Pi transporters in four barley genotypes differing in PAE. The results showed that there was no clear pattern in the expression of four paralogs of the high-affinity Pi transporter HvPHT1;1 among the four barley genotypes, but the expression of a low-affinity Pi transporter, HvPHT1;6, and its close homolog HvHPT1;3 was correlated with the genotypes differing in PUE. Interestingly, the expression of HvPHT1;6 and HvPHT1;3 was correlated with the expression of HvIPS1 (for P starvation inducible; noncoding RNA) but not with HvIPS2, suggesting that HvIPS1 plays a distinct role in the regulation of the low-affinity Pi transporters. In addition, high PUE was found to be associated with high root-shoot ratios in low-P conditions, indicating that high carbohydrate partitioning into roots occurs simultaneously with high PUE. However, high PUE accompanying high carbon partitioning into roots could result in low PAE. Therefore, the optimization of PUE through the modification of low-affinity Pi transporter expression may assist further improvement of PAE for low-input agriculture systems.