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• Long-term perennial grasses were compared in two locations and four growing seasons. • The main driving factor of bioethanol production was the biomass yield. • Optimization of harvest time allowed to gain more ethanol per unit mass. • Giant reed was the most chemically consistent feedstock among study factors. • Bioethanol biorefineries may gain fourfold the energy invested. Advanced biofuel production requires feedstock with specific composition in terms of quantity and quality. Decisions about the harvest time of perennial energy grasses have important implications for bioconversion process as well as for the life-span of the plant stand. Four-year data from long term-field trials were used to identify the optimal crop-specific harvest date (autumn vs. winter), in relation to advanced biofuel production. Three different perennial energy grasses (i.e., switchgrass, giant reed and miscanthus) were grown at Bologna and Catania (Italy), under northern and southern Mediterranean climate, respectively, adopting a low input management. In addition to the biomass yield and composition, the net energy value (NEV) from agricultural activities, and the energy return on investment (EROI) at the biorefinery gate was also evaluated. At Bologna, switchgrass biomass yield and quality (hemicellulose, cellulose and acid detergent lignin – ADL) were significantly enhanced when harvested in winter. At Catania, giant reed productivity, cellulose and ADL content were unaffected by harvest date, while hemicellulose and ash content decreased in winter. Biomass composition of miscanthus was more stable in Catania than Bologna, however, yield and components resulted significantly higher in Bologna than Catania, due mainly to the more even precipitation distribution throughout the vegetative crop development. The NEV was positive for all energy crops, treatments and locations, with the highest value for miscanthus grown at Bologna. The EROI of lignocellulosic bioethanol ranged from 4.16 for switchgrass in the autumn harvest at Bologna to 4.37 for miscanthus grown at Catania, which showed also the highest theoretical bioethanol production (169.2 kg h−1) at a feeding rate of 300 kg h−1 of raw material. Nonetheless, miscanthus grown at Bologna attained the highest bioethanol yield (12,254 L ha−1). This study proved that investigated perennial grasses are worth to be grown as lignocellulosic feedstock under low input for at least one decade, upon which Mediterranean region biorefineries may gain fourfold the energy invested. [ABSTRACT FROM AUTHOR]