Anaerobic sludge from palm oil mill effluent (POME) treatment plant was used as a source of inocula for the conversion of POME into hydrogen. Optimization of temperature and initial pH for biohydrogen production from POME was investigated by response surface methodology. Temperature of 60ºC and initial pHof 5.5 was optimized for anaerobic microflora which gave a maximum hydrogen production of 4820 ml H₂/l-POME corresponding to hydrogen yield of 243 ml H₂/g-sugar. Total sugar consumption and chemical oxygen demand (COD) removal efficiency were 98.7% and 46%, respectively. Long-term hydrogen production in continuous reactor at HRT of 2 days, 1 day and 12 hrs were 4850 ± 90, 4660 ± 99 and 2590 ± 120 ml H₂/l-POME, respectively. Phylogenetic analysis of the mixed culture revealed that members involved hydrogen producers in both batch and continuous reactors were phylogenetically related to the Thermoanaerobacterium thermosaccharolyticum. Batch reactor showed more diversity of microorganisms than continuous reactor. Microbial community structure of batch reactor was comprised of T. thermosaccharolyticum, T. bryantii, Thermoanaerobacterium sp., Clostridium thermopalmarium and Clostridium NS5-4, while continuous reactor was comprised of T. thermosaccharolyticum, T. bryantii and Thermoanaerobacterium sp. POME is good substrate for biohydrogen production under thermophilic condition with Thermoanaerobacterium species play an important role in hydrogen fermentation.