<P><B>Abstract</B></P> <P>Salt-containing wastes have increased in abundance in recent years. In this study, we investigated hydrogen production from intensive shrimp mariculture organic waste in batch culture experiments. Sludge samples were pretreated at different temperatures (50–110 °C) and for various time periods (10–120 min) to enrich the hydrogen-producing microflora. The results showed that all of the thermal pretreatments achieved higher hydrogen yields compared with those obtained in the experiments without heat treatment. Pretreatment at 70 °C achieved the maximum hydrogen yield. Different heat pretreatment time periods between 30 and 120 min had little influence on the hydrogen yield. Considering the energy consumption of the pretreatment process, 30 min at 70 °C were selected as the optimum pretreatment conditions. Microbial community DNA analysis and the diversity of the hydA gene showed that the number of bacterial species and the hydA gene diversity decreased as the pretreatment temperature increased.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Heat pretreatment was used to acquire hydrogen producing microflora. </LI> <LI> Heat pretreated 30 min at 70 °C were selected as the optimum pretreatment conditions. </LI> <LI> Bacterial species decreased as the pretreatment temperature increased. </LI> <LI> The hydA gene diversity decreased as the pretreatment temperature increased. </LI> </UL> </P>