Transcriptional activation of subfamily II PHT1 members in roots is associated with the enhanced phosphorus use efficiency and growth promotion of barley seedlings inoculated with Glomus species. The arbuscular mycorrhizal (AM) fungi symbiotic associations in cereal crops are known to regulate growth in cultivar-specific manner and induce phosphate (Pi) transporters (PHT1) in roots. In the present study, we observed that both AM colonization of roots by Glomus species and phosphate starvation enhanced phosphorus use efficiency (PUE) in barley seedlings. Our search for the full complement of PHT1 members in the recently sequenced barley genome identified six additional genes, totaling their number to 17. Both AM colonization and Pi starvation triggered activation of common as well as different PHT1s. Pi starvation led to the robust upregulation of HvPHT1;6.2/6.3 at 7d and weak activation of HvPHT1;1 in shoots at 3d time-point. In roots, only HvPHT1;1, HvPHT1;6.2/6.3, HvPHT1;7, HvPHT1;8, HvPHT1;11.2 and HvPHT12 were induced at least one of the time-points. AM colonization specifically upregulated HvPHT1;11, HvPHT1;11.2, HvPHT1;12 and HvPHT1;13.1/13.2, members belonging to subfamily II, in roots. Sucrose availability seems to be obligatory for the robust activation of HvPHT1;1 as unavailability of this metabolite generally weakened its upregulation under Pi starvation. Intriguingly, lack of sucrose supply also led to induction of HvPHT1;5, HvPHT1;8, and HvPHT1;11.2 in either roots or shoot or both. The mRNA levels of HvPHT1;5 and HvPHT1;11.2 were not severely affected under combined deficiency of Pi and sucrose. Taken together, this study not only identify additional PHT1 members in barley, but also ascertain their AM, Pi and sucrose-specific transcript accumulation. The beneficial role of AM fungi in the promotion of PUE and barley seedlings' growth is also demonstrated.