ABSTRACT Most cytology reports on ectomycorrhiza (ECM) have been less focused on the fungal structures, especially on the exploratory organ. This study aimed to evaluate the morphological response of explorative mycelia of ECM forming fungi with and without the occurrence of the ECM host. We assessed the mycelial aggregates of Rhizopogon roseolus which was inoculated with and without Pinus thunbergii under controlled laboratory conditions. The mycelial aggregates with the host produced strikingly complex hyphal strands relative to those without host. Light microscopy revealed that the cytology and plectology of both mycelial aggregates had the approximately similar architecture. The tubular hyphae diameter without the host was consistently larger than that with the host. This study confirmed that the septa diameter of the tubular hyphae conjunction of mycelial aggregates with the host were shorter than those without the host. In addition, partially and completely dissolved septa of the tubular hyphae were evident with and without host. We also described the thromboplerous hyphae, which have rarely been reported in vitro. These hyphae were produced in higher numbers near the fungal inoculum with and without the host. The current findings will add to the current knowledge regarding ECM fungi mycelial aggregate response to the host.