Recently, there have been global movements toward reducing the use of fossil resources as source of energy due to continuous depletion of petroleum fuel-reserves besides contributing to environmental problems such as greenhouse effect, global warming and climate change. Isobutanol is one of biomass-based fuels that has been recognizes for its potentiality as fuel additive or substitute due to its attractive physical properties. This paper investigates the production of isobutanol and alcohol tolerance by five different types of yeast (Saccharomyces cerevisiae,Kluyveromyces lactisGG799 andPichia pastorisKM71H, GS115 and X33) in batch fermentation. Based on the result obtained,P.pastorisX33 produced the highest concentration of isobutanol at 65 mg/l followed byP.pastorisGS115,K.lactisGG799,P.pastorisKM71H andS.cerevisiaewith concentration of 57 mg/l, 49 mg/l 49 mg/l and 46 mg/l respectively. This result proves that yeast is able to produce isobutanol naturally.S. cerevisiaehas been proven as good yeast in alcohol tolerance as it was capable to grow in more than 2% isobutanol and butanol of up to 2%. Among the different alcohols tested for alcohol tolerance, 3-methyl-1-butanol has the highest toxicity towards yeast growth as compared to isobutanol and butanol.