Hydrogen (H-2) is considered one of the most promising fuels for sustainable energy. Because nitrogenase produces H-2 as a normal by-product, we tested the N-2-fixing bacterial strains Burkholderia unamae and Burkholderia tropica to determine their H-2 production capacities. To maximize H-2 production, several culture conditions were tested and optimized, including atmospheric conditions, carbon sources and chemical compounds such as enzyme cofactors and sugar cane molasses. The results showed that both strains were capable of H-2 production. The culture medium with the highest H-2 yield was composed of 1% v/v molasses enriched with Na2MoO4 (0.2 g/L), FeSO4 (0.2 g/L) and cysteine (0.02 g/L) under a partial vacuum (air 20% v/v) without Ar final atmosphere. Under these conditions, the highest H-2 production rate obtained was 24.64 mmol H-2/L for B. unamae. The present study contributes an optimization process for H-2 production in N-2-fixing Burkholderia species. We propose further research and development to improve H-2 production rates in order to make biohydrogen a tangible reality. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.