Aureobasidium pullulans, popularly known as black yeast, is one of the most widespread saprophytic fungus associated with wide range of terrestrial and aquatic habitats. The fungus has widely been employed in production of an economically important polysaccharide pullulan. Total 50 isolates of Aureobasidium pullulans were isolated from different flowers and leaves samples, out of which 10 thermotolerant strains produced pullulan. One thermotolerant non-melanin pullulan producing strain, designated as RS-4, produced highest pullulan (6.5±0.02 g/100ml) at 37oC, pH 5.0 in 72h of incubation with 5% sucrose and 0.5% yeast extract in a non-stirred flask fermentation system with working volume 100ml.