Lactic acid was produced by simultaneous saccharification and fermentation (SSF) of liquid hot water (LHW)-pretreated and non-LHW-pretreated alfalfa fibers. The Lactobacillus plantarum and L. delbrueckii strains produced 0.464 and 0.354 g of lactic acid per g of dry matter of alfalfa fiber, respectively, by non-LHW pretreatment. L. xylosus and L. pentoaceticus produced lower yields of lactic acid from the same amount of alfalfa fiber, however, their acetic acid production was higher. These Lactobacillus strains did not require any additional nutrients during SSF of non-LHW-pretreated alfalfa fiber. After LHW pretreatment, the "raffinate" cellulosic fraction of alfalfa required additional nutrients for lactic acid production by SSF. Both L. plantarum and L. delbrueckii produced 0.606 and 0.59 g of lactic acid per g of dry matter of fiber, respectively. However, the "extract" soluble hemicellulosic fraction of alfalfa produced 0.38 to 0.62 g of lactic acid per g of dry matter extract during SSF and did not require nutrient supplementation. These results suggest that during the LHW pretreatment, alfalfa fiber nutrients are lost in cellulosic fractions but retained in hemicellulosic extract fractions.