•δ56Fe heterogeneity is observed in plume-influenced basalts from the GSC.•The δ56Fe composition of these basalts cannot result from melting of a peridotite.•Lithological heterogeneity in the Galápagos mantle plume is required.•δ56Fe heterogeneity of oceanic basalts results from lithological heterogeneity. The extent of lithological heterogeneity in the Earth's convecting mantle is highly debated. Whilst the presence of pyroxenite in the mantle source regions of Ocean Island Basalts (OIBs) has traditionally been constrained using the minor-element chemistry of olivine phenocrysts, recent studies have shown that the Ni and Mn contents of primitive olivines are influenced by the conditions of mantle melting, as well as magma chamber processes. Nevertheless, constraining the lithological properties of the mantle is important due to its influence on the P-T path followed by solid mantle material during adiabatic ascent, as well as the density of upwelling mantle plumes. We have therefore explored the use of Fe-isotopes as a novel method of tracing lithological heterogeneity in the mantle source regions beneath plume-influenced segments of the global Mid-Ocean Ridge system as well as OIBs. We present new Fe-isotope (δ56Fe) and trace-element data for 26 basaltic glasses from the plume-influenced Galápagos Spreading Centre to investigate the relative roles of pyroxenite and peridotite in the mantle source region of oceanic basalts. Our data reveals significant heterogeneity in the Fe-isotope composition of the Galápagos Spreading Centre basalts (+0.05 to +0.25‰ δ56Fe), which correlates with key major- and trace-element parameters (e.g. CaO(8)/Al2O3(8), La/Smn). Application of new models developed to calculate Fe-isotope fractionation during mantle melting, alongside Monte Carlo simulations for melting of a 2-component peridotite mantle, show that this variation cannot be caused by changes in melting processes and/or oxygen fugacity of a peridotitic mantle. Instead, our new δ56Fe data is best explained by variations in the proportion of isotopically-heavy pyroxenite-derived melt that contributes to the GSC basalts, and conclusively shows that lithological heterogeneity exists in the Galápagos mantle plume. Our findings have implications for the moderately-heavy δ56Fe compositions measured in plume-influenced basalts from the Society Islands, Rochambeau Ridges of the Lau back-arc basin, and the FAMOUS segment of the Mid-Atlantic Ridge, which we suggest may also represent contribution from pyroxenite-derived melts.