Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses by improving membrane structure and scavenging reactive oxygen species. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene emitting (IE) and non-isoprene emitting (NE) poplars. We demonstrated that the total amount of mono- (MGDG), di-galactosyldiacylglycerols (DGDG), phospholipids (PL), and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid (18:3) in NE chloroplasts was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects PSII photochemical efficiency (ΦPSII). The low ΦPSII in NE plants was negatively correlated with non-photochemical quenching (NPQ) and the energy-dependent (qE) component of NPQ. Transmission electron microscopy (TEM) revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained less grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in isoprene-emitting species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes.