The thermotolerant Rhizopus microsporus DMKU 33 capable of producing L-lactic acid from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and growth temperature and pH ranges. The concentrations of (NH4)(2)SO4, ICH2PO4, MgSO4 and ZnSO4 center dot 7H(2)O in the fermentation medium was optimized for lactic acid production from liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake -flasks at 40 degrees C. The fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH 5.5 in 3 days. Lactic acid production was further increased to 105-118 g/L with a yield of 0.93 g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus advantageous to use in simultaneous saccharification and fermentation for L-lactic acid production from low-cost starchy substrates.