Lactic acid bacteria (LAB) were isolated from fresh sugar cane juice. It was found that several isolates exhibited a clear zone and growth on deMan, Rogosa, Sharpe (MRS) agar supplemented with sodium azide, bromocresol purple and sucrose. However, only 17 isolates which formed large yellow areas were selected for further investigations. These isolates were further identified according to their morphological and biochemical characteristics. It was found that 10 of these isolates were homofermenters. One of these 10 isolates was selected for mutagenesis using chemical (Ethidium bromide) and physical (UV-B) mutagens followed by biochemical characterisation. A total of 112 mutants were isolated and 9 homofermentative isolates were further investigated for their ability to produce lactic acid. 1H-NMR spectroscopy confirmed that all mutant isolates produced lactic acid as the sole fermentation product