Previous studies have demonstrated the feasibility of employing by-products of the olive and wine sectors for the production of Pleurotus mushrooms with enhanced functionalities. In this work we investigated the influence of endogenous and exogenous factors on free amino acids (FAAs) profile of Pleurotus ostreatus, P. eryngii and P. nebrodensis mushrooms produced on wheat straw (WS), alone or mixed with grape marc (GM), and on by-products of the olive industry (OL). Overall, 22 FAAs were determined in substrates and mushrooms, including all the essential amino acids, the neurotransmitter γ-aminobutyric acid (GABA) and ornithine. On a dry weight (dw) basis, total FAAs ranged from 17.37 mg/g in P. nebrodensis to 130.12 mg/g in P. ostreatus samples, with alanine, leucine, glutamine, valine and serine predominating. Similar distribution patterns were followed by the monosodium glutamate (MSG)-like, sweet and bitter FAAs. Significant differences in FAAs level were observed among the species examined and among the cultivation substrates used. Principal Component Analysis (PCA) performed on the entire FAAs profile of six Pleurotus strains, clearly separated P. ostreatus from P. eryngii and P. nebrodensis, in accordance to their phylogenetic affinity. This is the first report of FAAs in P. nebrodensis.