Biohydrogen production is a cheap and clean way to obtain hydrogen gas. In subtropical countries such as Brazil the average temperatures of 27 C can favor the hydrogen producing bacteria growth. A mixed culture was obtained from a subtropical sludge treating brewery wastewater and anaerobic batch reactors were fed with glucose, sucrose, fructose and xylose in low concentrations (2.0, 5.0 and 10.0 g L À1) at 37 C, initial pH 5.5 and headspace with N 2 (99%) to maintain the anaerobic conditions. The inoculum was a subtropical granulated sludge from UASB (Upflow Anaerobic Sludge Blanket) reactor treating brewery wastewater. The higher H 2 yields were obtained in reactors operated with 2 and 5 g L À1 of fructose and they were 1.5 mol H 2 mol À1 of fructose and 1.3 mol H 2 mol À1 of sucrose, respectively. The volatile fatty acids (VFA) generated at the end of operation were, predominantly, butyric and acetic acid, indicating the favoring of the metabolic route of hydrogen generation by the consortium of anaerobic bacteria from the brewery wastewater. Biomolecular analyses revealed the predominance of hydrogen producing bacteria from Firmicutes phylum distributed in the families Streptococcaceae, Veillonellaceae and uncultured bacteria. These results confirm future applications of subtropical sludges with agroindustrial wastewaters containing low concentrations of sugars on hydrogen generation.