The bio-hydrogen generation potential of sugar industry wastes was investigated. In the first part of the study, acidogenic anaerobic culture was enriched from the mixed anaerobic culture (MAC) through acidification of glucose. In the second part of the study, glucose acclimated acidogenic seed was used, along with the indigenous microorganisms, MAC, 2-bromoethanesulfonate treated MAC and heat treated MAC. Two different COD levels (4.5 and 30g/L COD) were investigated for each culture type. Reactors with initial COD concentration of 4.5g/L had higher H2 yields (20.3-87.7mL H2/g COD) than the reactors with initial COD concentration of 30g/L (0.9-16.6mL H2/g COD). The 2-bromoethanesulfonate and heat treatment of MAC inhibited the methanogenic activity, but did not increase the H2 production yield. The maximum H2 production (87.7mL H2/g COD) and minimum methanogenic activity were observed in the unseeded reactor with 4.5g/L of initial COD.