Inorganic polyphosphate (poly P) is a linear polymer of phosphoanhydride-linked phosphate residues that occurs in all organisms and cells. It was found in all organelles and is particularly abundant in fungal vacuoles. The fungal cell wall also contains poly P, but very little is known about the nature and functions of poly P in this compartment. Here, we describe a novel method for the specific quantification and visualization of poly P in fungal cell walls. Selective extraction in high salt buffer revealed large poly P stores in cell walls of Mucorales and lower amounts in most other fungi tested. Staining with specific poly P binding proteins (PBPs) enabled the visualization of poly P in cell walls of selected species from all fungal phyla. The presence of an extracellular phosphate pool in the form of a strongly negatively charged polymer is suggested to have important functions as a phosphate source in mycorrhizal interactions, an antimicrobial compound or protection against toxicity of heavy metals.