Many ectomycorrhizal fungi (EMF) are generalists, but most plant genera that form ectomycorrhizas have at least some fungal partners that are specific to that host genus. Because shared mycorrhizal fungi mediate plant community interactions, host preference has implications for plant succession and competition. We studied the EMF of oaks (Quercus spp.) and pines (Pinus spp.) in a forest in northern Florida, USA, focusing on symbionts shared with longleaf pine (Pinus palustris Mill.). Longleaf pine is an important species in the southeastern USA, both for timber plantations and for restoring savanna and woodland habitat. However, we found no research on the composition of naturally occurring EMF on longleaf pine roots. A lower proportion of EMF operational taxonomic units (OTUs) were found colonizing both oaks and pines than expected, providing evidence of host preference within the community. Although most EMF were detected only on either oaks or pines, the OTUs found on both tended to be frequently occurring and abundant. Cenococcum OTUs were found to be significantly associated with oaks, an unexpected finding as this genus is widespread, with a broad host range. These results suggest that host preference of EMF may structure EMF communities and therefore influence ecosystem effects of mycorrhizal networks.