Root endophytic fungi are seen as promising alternatives to replace chemical fertilizers and pesticides in sustainable and organic agriculture systems. Fungal endophytes structure formations play key roles in symbiotic intracellular association with plant-roots. To compare the morphologies of Ascomycete endophytic fungi in wheat, we analyzed growth morphologies during endophytic development of hyphae within the cortex of living vs. dead root cells. Confocal laser scanning microscopy (CLSM) was used to characterize fungal cell morphology within lactofuchsin-stained roots. Cell form regularity Ireg and cell growth direction Idir, indexes were used to quantify changes in fungal morphology. Endophyte fungi in living roots had a variable Ireg and Idir values, low colonization abundance and patchy colonization patterns, whereas the same endophyte species in dead (gamma-irradiated) roots had consistent form of cells and mostly grew parallel to the root axis. Knot, coil and vesicle structures dominated in living roots, as putative symbiotic functional organs. Finally, an increased hypha septation in living roots might indicate local specialization within endophytic Ascomycota. Our results suggested that the applied method could be expanded to other septate fungal symbionts (e.g. Basidiomycota). The latter is discussed in light of our results and other recent discoveries.