The present work deals with the improvement of multiple stress tolerance in a glucose xylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L-1 furfural and 8 g L-1 acetic acid. The mutant also showed good growth at high temperature (39-40 degrees C). A study on the combined effect of multiple stresses during fermentation of glucose-xylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39 degrees C) and inhibitor stress (0.25 g L-1 vanillin, 0.5 g L-1 furfural and 4 g L-1 acetic acid), the mutant produced ethanol with a yield of 0.379 g g(-1), while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g(-1). Further, under the synergistic effect of sugar (250 g L-1), thermal (39 degrees C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L-1 ethanol by utilizing 162.42 g L-1 of glucose xylose mixture giving an ethanol yield of 0.295 g g(-1) and productivity of 0.57 g L-1 h(-1). Under same condition the fusant RPR39 produced a maximum of 30.0 g L-1 ethanol giving a yield and productivity of 0.21 g g(-1) and 0.42 g L-1 h(-1) respectively. The molecular characterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.