The ability of three Australian Pisolithus species, discriminated on the basis of ITS sequence data, to utilize a range of inorganic and organic nitrogen sources was assessed in liquid axenic culture. Both intra-, and putative interspecific, variation in nitrogen source utilization was observed. Most isolates demonstrated a preference for NH4+ over NO3−, although some showed no significant preference for either inorganic source. All isolates utilized a range of amino acids. Species I isolates demonstrated a preference for acidic and/or neutral amino acids over basic acids, while species II and III isolates generally utilized amino acids poorly relative to species I. Although most isolates utilized BSA poorly, two species I isolates that had been maintained in axenic culture for > 10 y grew well on this substrate, suggesting possible changes in nitrogen utilization with extended storage in axenic culture.