Oyster mushroom (Pleurotus ostreatus) is still one of the most cultivated edible and medicinal mushrooms. Despite its frequent cultivation around the world, there is currently just a little information available on the variability of strains in terms of the content of β-glucans in them. This work presents an extensive study of 60 strains in terms of the content of α-glucans and β-glucans in their caps and stipes. The authenticity of the production strains based on an analysis of the variability of their genome by CDDP (Conserved DNA-derived polymorphism) markers was confirmed, whereas identical CDDP profiles were identified between samples 45, 89, 95, and 96. Genetic variability of the analyzed production strains showed a high polymorphism and effective discriminative power of the used marking technique. Medium positive correlations were found among the CDDP profiles and β-glucan content in the group of strains that generated the same CDDP profiles, and low negative correlation was found among these profiles in the group of low β-glucan content strains. For the determination of glucans content, Mushroom and Yeast analytical enzymatic kit (Megazyme, Bray, Co. Wicklow, Ireland) were used. The results clearly showed that the stipe contains on average 33% more β-glucans than the cap. The minimum detected β-glucan content in the stipe was in strain no. 72, specifically 22%, and the maximum in strain no. 43, specifically 56%, which after the conversion represents a difference of 155%. From the point of view of β-glucan content, the stated strain no. 43 appears to be very suitable for the commercial production of β-glucans under certain conditions.