Dark fermentation shares many features with anaerobic digestion with the exception that to maximize hydrogen production, methanogens and hydrogen-consuming bacteria should be inhibited. Heat treatment is widely applied as an inoculum pre-treatment due to its effectiveness in inhibiting methanogenic microflora but it may not exclusively select for hydrogen-producing bacteria. This work evaluated the effects of heat treatment on microbial viability and structure of anaerobic granular sludge. Heat treatment was carried out on granular sludge at 100 °C with four residence times (0.5, 1, 2 and 4 h). Hydrogen production of treated sludges was studied from glucose by means of batch test at different pH values. Results indicated that each heat treatment strongly influenced the granular sludge resulting in microbial communities having different hydrogen productions. The highest hydrogen yields (2.14 moles of hydrogen per mole of glucose) were obtained at pH 5.5 using the sludge treated for 4 h characterized by the lowest CFU concentration (2.3 × 10(3)CFU/g sludge). This study demonstrated that heat treatment should be carefully defined according to the structure of the sludge microbial community, allowing the selection of highly efficient hydrogen-producing microbes.