The ecological and evolutionary outcomes of plant–fungal interactions are strongly influenced by genome size and ploidy, yet the ploidy level of both partners is rarely assessed simultaneously. Epichloë symbioses with Pooideae grasses are established model systems for exploring these dynamics, but associations between polyploid hosts and haploid endophytes remain poorly documented. In this study, the association of the Iberian endemic Festuca rothmaleri —which includes tetraploid, hexaploid, and octoploid cytotypes—with Epichloë fungal endophytes is documented for the first time. An integrative, method-rich framework combining cytogenetics, morphometrics, and multilocus phylogenetics revealed a strikingly asymmetric interaction, with all cytotypes harboring a single haploid strain of Epichloë festucae . Two methodological innovations were developed: (i) an image-based tool for automated measurement of asexual structures, including the novel metric “conidial area,” and (ii) a flow cytometry protocol for estimating fungal genome size. Despite morphological variability, all fungal isolates shared similar genome sizes and formed a well-supported monophyletic lineage in a coalescent species tree based on nuclear loci sequences ( actG , CalM , ITS, tefA , tubB ). This work provides the first comprehensive characterization of a haploid Epichloë endophyte spanning multiple naturally distributed host ploidy levels and highlights a rare but promising system for future evolutionary, physiological, and ecological studies of plant–fungal interactions.