Estimations of genome size and its variation can provide valuable information regarding the genetic diversity of organisms and their adaptation potential to heterogeneous environments. We used flow cytometry to characterize the variation in genome size among 40 isolates of Cenococcum geophilum, an ectomycorrhizal fungus with a wide ecological and geographical distribution, obtained from two serpentine and two non-serpentine sites in Portugal. Besides determining the genome size and its intraspecies variation, we wanted to assess whether a relationship exists between genome size and the edaphic background of the C. geophilum isolates. Our results reveal C. geophilum to have one of the largest genome sizes so far measured in the Ascomycota, with a mean haploid genome size estimate of 0.208 pg (203 Mbp). However, no relationship was found between genome size and the edaphic background of the sampled isolates, indicating genetic and demographic processes to be more important for shaping the genome size variation in this species than environmental selection. The detection of variation in ploidy level among our isolates, including a single individual with both presumed haploid and diploid nuclei, provides supportive evidence for a possible cryptic sexual or parasexual cycle in C. geophilum (although other mechanisms may have caused this variation). The existence of such a cycle would have wide significance, explaining the high levels of genetic diversity and likelihood of recombination previously reported in this species, and adds to the increasing number of studies suggesting sexual cycles in previously assumed asexual fungi.