There are several parameters that affect the hydrogen (H-2) production when using organic solid waste in a discontinuous process, such as the hydraulic retention time (HRT). To optimize the process, it is necessary to determine the HRT such that the H-2 production is maximized. The objective of this study is to evaluate the effect of different HRTs on the hydrogen production in an anaerobic sequencing batch reactor (ASBR) using food waste as the substrate. The microbial community was analyzed and correlated with the reactor performance. The results showed that the highest percentage of H-2 in biogas, maximum volumetric H-2 production, and maximum H-2 production rate was obtained with an HRT of 24 h. The percentage of H-2 in biogas varied from 22 to 53%, which depended on the HRT. Acetic acid was the main volatile fatty acid obtained. The highest propionic acid production was observed with an HRT of 6 h, which was related to a decrease in H-2 generation, where a low diversity and low evenness were obtained compared with those of the reactor samples obtained from different HRTs. The genus Megasphaera was found to be the dominant genus in the microbial community. Copyright (c) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.