The barley cultivar Sarab 1 (SRB1) can continue photosynthesis despite its low Fe acquisition potential via roots and dramatically reduced amounts of photosystem I (PSI) reaction-center proteins under Fe-deficient conditions. We compared the characteristics of photosynthetic electron transfer (ET), thylakoid ultrastructure, and Fe and protein distribution on thylakoid membranes among barley cultivars. The Fe-deficient SRB1 had a large proportion of functional PSI proteins by avoiding P700 over-reduction. An analysis of the thylakoid ultrastructure clarified that SRB1 had a larger proportion of non-appressed thylakoid membranes than those in another Fe-tolerant cultivar, Ehimehadaka-1 (EHM1). Separating thylakoids by differential centrifugation further revealed that the Fe-deficient SRB1 had increased amounts of low/light-density thylakoids with increased Fe and light-harvesting complex II (LHCII) than did EHM1. LHCII with uncommon localization probably prevents excessive ET from PSII leading to elevated NPQ and lower PSI photodamage in SRB1 than in EHM1, as supported by increased Y(NPQ) and Y(ND) in the Fe-deficient SRB1. Unlike this strategy, EHM1 may preferentially supply Fe cofactors to PSI, thereby exploiting more surplus reaction center proteins than SRB1 under Fe-deficient conditions. In summary, SRB1 and EHM1 support PSI through different mechanisms during Fe deficiency, suggesting that barley species have multiple strategies for acclimating photosynthetic apparatus to Fe deficiency.