Suillus luteus is the most important wild edible ectomycorrhizal mushroom harvested and exported in Chile. This introduced species forms mycorrhizal symbiosis with Pinus radiata, the most important exotic forest tree. To obtain optimized protocols for the controlled infection of P. radiata trees with this species, it is important to determine the appropriate culture conditions. Therefore, we studied the effect of the geographical origin (three localities from the Maule Region of Chile), culture media (Modified Melin and Norkrans (MMN) and Malt Extract Agar (MEA) 2%), and pH (5.0, 5.8, and 6.5) on the in vitro growth dynamics of S. luteus over 37 days. The results showed significant differences in the growth rate and colony diameter as a consequence of its geographical origin. However, no differences were found for either pH or culture medium. Our results confirm that a non-specific, economical culture medium, such as MEA 2%, may be used to obtain a suitable amount of mycelium for both medium- and large-scale assays of inoculation with this species.