Novel strains selected from various genera of yeasts were investigated with regard to pyruvate productivity from glucose. The ability to produce pyruvate was found in yeasts belonging to the genera Saccharomyces, Toluropsis, Pichia, Candida and Trichosporon with a new resting cell method using the thiamine analogue, oxythiamine. In this first screening, strains belonging to the genus Saccharomyces showed the highest pyruvate productivity. In secondary screening using a fermentative method, strains belonging to the genus Torulopsis showed higher pyruvate productivity, with Torulopsis glabrata exhibiting the highest fermentative pyruvate productivity. These pyruvate producing yeast strains were divided into two types. The first type of yeast showed a high accumulation of pyruvate with the resting cell method, but a low accumulation with the fermentative method. The second type of yeast showed a high accumulation of pyruvate with the fermentative method, but a low accumulation with the resting cell method. The first type did not commonly possess thiamine-auxotrophy, while the second type exhibited at least double auxotrophy for both biotin and thiamine. All six strains belonging to T. glabrata required four vitamins; nicotinic acid, thiamine, pyridoxine and biotin for their growth and accumulated a high conversion of pyruvate (53.7 mM-300 mM) under the fermentative screening conditions. T. glabrata IFO 0005 was selected as the best strain among the T. glabrata. The optimum concentrations of these vitamins for the highest fermentative pyruvate productivity by T. glabrata IFO 0005 were determined. Thiamine and nicotinate were more effective towards productivity than other vitamins. The accumulation of pyruvic acid reached a final concentration of 648 mM (57.0 g/l as free pyruvic acid) at 59 h from an initial glucose concentration of 100 g/l by T. glabrata IFO 0005 under the optimal vitamin concentrations determined in this study. Consequently, the auxotrophy of multiple vitamins which are involved in the metabolism of pyruvate proved to be highly effective in the fermentative production of pyruvate from glucose.