>Magister Scientiae - MSc / The depletion of oil reserves and the constant discharge of greenhouse gasses (GHG) that are associated with global warming have forced both political and scientific sectors to pursue alternative, renewable and sustainable fuels that will be blended with petrol and ultimately replace it as the fuel of choice. Bioethanol is a form of fuel that is obtained from natural materials such as biomass. Starch and sugar containing materials are the primary carbon sources for bioethanol production and a range of feedstocks are currently being exploited for this purpose worldwide.This study was aimed at measuring, comparing and analyzing fermentable sugars liberated by sorghum and three other grain crops (maize, barley and wheat) that are grown in South Africa and subsequently analyze ethanol yield after fermentation. Starch was extracted from sorghum, maize, barley and wheat via hot water treatment and hydrolyzed by use of !-amylase, gluco-amylase and a cocktail of both enzymes under various conditions to determine optimum hydrolysis conditions. The resultant liberated soluble sugars were measured with a pocket refractometer and High Performance Liquid Chromatography (HPLC) respectively. Hydrolysates obtained under optimum conditions were fermented with various ethanol producing microbial strains and a high-performing strain was selected. The selected high-performing strain (Saccharomyces cerevisiae NT 53) was used to ferment different grain hydrolysates (sorghum, maize, barley and wheat).The working volumes of the solutions were increased ten-fold (small-scale) and experiments were performed using sorghum grains as substrates and alcohol content was measured with an Alcolyzer Wine M instrument. The optimum hydrolysis conditions for the grain crops were determined and it was found that the enzymes performed well at 70°C and starch was hydrolyzed within the first hour.Sixty grams per litre (60 g/L) of grain solution produced a maximum of 50.8 g/L of glucose when treated with the cocktail treatment. However gluco-amylase facilitated a similar production, at 47.8 g/L glucose. Sorghum and maize produced high glucose amounts and subsequent ethanol amounts, and maximum fermentation efficiencies of 87 % and 98 % respectively when fermented with the high performing NT 53 strain. The NT 53 strain was compared with commercial baker’s yeast and they yielded similar ethanol amounts across the grain types. Under small-scale conditions, sorghum retained the consistency of yielding similar glucose amounts compared to laboratory-scale (50ml) conditions and when analyzed with the Alcolyzer, sorghum yielded a maximum alcohol content of approximately 2 % v/v. This study also showed that gluco-amylase alone was sufficient for starch hydrolysis and sorghum a more favourable and less expensive crop for ethanol production in South Africa.