Molasses "B" is a rich co-product of the sugarcane process. It is obtained from the second step of crystallization and is richer in fermentable sugars (50-65%) than the final molasses, with a lower non-sugar solid content (18-33%); this co-product also contains good vitamin and mineral levels. The use of molasses "B" for ethanol production could be a good option for the sugarcane industry when cane sugar prices diminish in the market. In a complex medium like molasses, osmotolerance is a desirable characteristic for ethanol producing strains. The aim of this work was to evaluate the use of molasses "B" for ethanol production using Saccharomyces cerevisiae ITV-01 (a wild-type yeast isolated from sugarcane molasses) using different initial sugar concentrations (70-291 g L(-1)), two inoculum sizes and the addition of nutrients such as yeast extract, urea, and ammonium sulphate to the culture medium. The results obtained showed that the strain was able to grow at 291 g L(-1) total sugars in molasses "B" medium; the addition of nutrients to the culture medium did not produce a statistically significant difference. This yeast exhibits high osmotolerance in this medium, producing high ethanol yields (0.41 g g(-1)). The best conditions for ethanol production were 220 g L(-1) initial total sugars in molasses "B" medium, pH 5.5, using an inoculum size of 6 × 10(6) cell mL(-1); ethanol production was 85 g L(-1), productivity 3.8 g L(-1 )h(-1) with 90% preserved cell viability.