The plant biomass and agro-industrial wastes show great potential for their use as attractive low cost substrates in biotechnological processes. Wheat straw and corn cob as hemicellulosic substrates were acid hydrolyzed and enzymatically saccharified for high xylose production. The hydrolysate was concentrated and fermented by using Saccharomyces cerevisiae and Kluyveromyces for production of xylitol. Acid hydrolysis of wheat straw and corn cob in combination with enzymatic hydrolysis showed great potential for production of free sugars from these substrates. Kluyveromyces produced maximum xylitol from acid treated wheat straw residues with enzymatic saccharification. The percentage xylitol yield was 89.807 g/L and volumetric productivity of 0.019 g/L/h. Kluyveromyces also produced maximum xylitol from corn cob acid hydrolyzed liquor with xylitol yield 87.716 g/L and volumetric productivity 0.018 g/L/h. Plant and agro-industrial biomass can be used as a carbohydrate source for the production of xylitol and ethanol after microbial fermentation. This study revealed that wheat straw acid and enzyme hydrolyzed residue proved to be best raw material for production of xylitol with S. cerevisiae. The xylitol produced can be utilized in pharmaceuticals after purification on industrial scale as pharmaceutical purposes.