The influence of genetic (species, strain) and environmental (substrate) factors on the volatile profiles of eight strains of Pleurotus eryngii and P. ostreatus mushrooms cultivated on wheat straw or substrates enriched with winery or olive oil by products was investigated by headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Selected samples were additionally roasted. More than 50 compounds were determined in fresh mushroom samples, with P. ostreatus presenting higher concentrations but a lower number of volatile compounds compared to P. eryngii. Roasting resulted in partial elimination of volatiles and the formation of pyrazines, Strecker aldehydes and sulfur compounds. Principal component analysis on the data obtained succeeded to discriminate among raw and cooked mushrooms as well as among Pleurotus species and strains, but not among different cultivation substrates. Ketones, alcohols and toluene were mainly responsible for discriminating among P. ostreatus strains while aldehydes and fatty acid methyl esters contributed more at separating P. eryngii strains.