The arbuscular mycorrhizal (AM) symbiosis is characterized by the reciprocal exchange of nutrients. AM fungi are oleaginous microorganisms that obtain essential fatty acids from host plants. A lipid biosynthesis and delivery pathway has been proposed to operate in inner root cortex cells hosting arbuscules, a cell type challenging to access microscopically. Despite the central role lipids play in the association, lipid distribution patterns during arbuscule development are currently unknown. We developed a simple co-staining method employing fluorophore-conjugated Wheat Germ Agglutinin (WGA) and a lipophilic blue fluorochrome, Ac-201, for the simultaneous imaging of arbuscules and lipids distributed within arbuscule-containing cells in high resolution. We observed lipid distribution patterns in wild-type root infection zones in a variety of plant species. In addition, we applied this methodology to mutants of the Lotus japonicus GRAS transcription factor RAM1 and the Oryza sativa half-size ABC transporter STR1, both proposed to be impaired in the symbiotic lipid biosynthesis-delivery pathway. We found that lipids accumulated in cortical cells hosting stunted arbuscules in Ljram1 and Osstr1, and observed lipids in the arbuscule body of Osstr1, suggesting that in the corresponding plant species, RAM1 and STR1 may not be essential for symbiotic lipid biosynthesis and transfer from arbuscule-containing cells, respectively. The versatility of this methodology has the potential to help elucidate key questions on the complex lipid dynamics fostering AM symbioses.