After entering the cell, the phosphorilation is the first chemical transformation that sugar endures. In yeast and higher organisms such reaction is mediated by ATP-dependent kinases. There are three enzymes in Saccharomyces cerevisae yeast which catalyze sugar phosphorilation: a glicokinase (that phosphorate glucose and manose) and two hexokinases (that phosphorate glucose, manose and fructose). In this work, Saccharomyces cerevisiae hexokinase-less strains were used in fermentations studies. These strains don’t have the hexokinases A and B and were obtained by crossing the strains Acc Y05867 and Acc Y14620 (with a blockage in the gene HXK1 and HXK2, respectivately, responsibles for the hexokinase enzyme A and B production), both obtained from EUROSCARF. After crossing, 23 strains were obtained, and then, three (U1, I1 and J3) were selected for presenting a smaller growth in YEPF. In fermentations with medium with fructose, this sugar didn’t consumed but, in media with glucose, this sugar were completely consumed and transformed into ethanol. In media containing sucrose (180.3 g.L^-1) in fermentation with cells recycles, it was well characterized the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where production of fructose (until 90g.L^-1) and ethanol (until 42.3g.L^-1) occurred in cycles of 12 hours, in which hexokinase-less yeasts performed high growth and viability rates after 9 consecutive cycles. When sugar cane juice and molasses was used as substrate the yeasts were able to ferment only into ethanol, with high efficiency, leaving fructose in medium.