The objective of this work was to comparatively evaluate the production of biohydrogen (bio-H-2) from tequila vinasses at optimized mesophilic and thermophilic conditions and to elucidate the main metabolic routes involved. Optimal temperatures of 35 degrees C and 55 degrees C, and pH of 5.5 maximized the bio-H 2 production rates, 25.5 +/- 0.01 NmL h(-1) and 169.9 +/- 8.9 NmL h(-1) in the mesophilic and thermophilic regimens, respectively. During the operation of anaerobic sequencing batch reactors, the thermophilic process allowed a volumetric bio-H-2 production rate of 519 +/- 13 NmL-H(2)L(-1)d(-1) equivalent to 750 +/- 19 NmL-H-2 L-vinasse(-1), while the mesophilic one 448 +/- 23 NmL-H-2 L(-1)d(-1) and 647 +/- 33 NmL-H-2 L-vinasse(-1), respectively. Furthermore, the gas produced under thermophilic conditions showed high hydrogen content (86.5%). Finally, formate degradation and glucose fermentation to acetic and butyric acids were the main metabolic routes involved in bio-H-2 production under thermophilic conditions, while at mesophilic conditions, the lactate and formate degradation pathways governed. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.