Ganoderma lingzhi (G. lingzhi), G. sinense, G. applanatum, etc. belongs to the Ganoderma genus of polypore mushrooms which contain rich polysaccharides valuable for nutrition and positive medicinal effects. In order to evaluate polysaccharide content in Ganoderma mycelia obtained in the fermentation process quickly and accurately, in this work we employed infrared spectroscopy to examine different Ganoderma stains of samples from diversified sources. Through mid-infrared (mid-IR) spectroscopy, we could identify the most relevant spectral bands required for polysaccharide evaluation, and through near-infrared (NIR) spectroscopy, we could establish the quantification model for making satisfactory prediction of polysaccharide ingredient content. As such, we have achieved an effective and convenient approach to quantitative assessment of the total polysaccharides in Ganoderma mycelia but also demonstrated that infrared spectroscopy can be a powerful tool for quality control of Ganoderma polysaccharides obtained from industrial production.