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This study aimed to produce biohydrogen from sugarcane vinasse in an anaerobic fluidized bed reactor operated at ambient temperature (26-35 °C) with a progressive increase of the organic load rate (OLR) (17-155 kg-COD m-3 d-1) and without influent pH control. Hydraulic retention times (HRT) of 12, 6 and 4 h were applied to increase the OLR. The highest hydrogen yield, hydrogen production rate and percentage of hydrogen in the biogas were obtained in the HRT of 4 h, corresponding to the values of 2.40 mol-H2 mol-1-carbohydrate, 0.33 L-H2 h-1 L-1-reactor, and 33%, respectively. The butyric acid fermentative route was favored in the process. The PCR/DGGE analysis showed that the microbial community structure was modified along operational stages and increasing OLR caused an increase in microbial diversity. The results indicate that biohydrogen production from anaerobic digestion of sugarcane vinasse can be achieved with no alkalization pretreatment.