Biohydrogen production via fermentative routes offers considerable advantages in waste recycling and sustainable energy production. This can be realized by single-stage dark or photofermentative processes, or by a two-stage integrated process; the latter offering the higher production yields due to complete conversion of sugar substrates into H-2 and CO2. However, problems arising from the integration of these two processes limit its scale-up and implementation. Hence, high efficiency one-step fermentative biohydrogen production processes from sugar-rich wastes are preferable. In this study, different strains of purple non-sulfur bacteria were investigated for their biohydrogen production capacity on pure sucrose and sugar beet molasses, and the feasibility of single-stage photofermentative biohydrogen production was evaluated. A single-stage photofermentation process was carried out using four different strains of purple non-sulfur bacteria (Rhodobacter capsulatus DSM 1710, R. capsulatus YO3, Rhodobacter sphaeroides O.U.001, and Rhodopseudomonas palustris DSM 127) on different initial sucrose concentrations. The highest hydrogen yield obtained was 10.5 mol H-2/mol of sucrose and the maximum hydrogen productivity was 0.78 mmol/L h by Rp. palustris on 5 mM sucrose. A hydrogen yield of 19 mol H-2/mol sucrose, which represents 79% of theoretical yield, and a maximum hydrogen productivity of 0.55 mmol/L h were obtained by Rp. palustris from sugar beet molasses. The yield was comparable to those values obtained in two-stage processes. The present study demonstrates that single-stage photofermentation using purple non-sulfur bacteria on sucrose-based wastes is promising.