Dark septate (DS) fungal endophytes are the primary root colonizers of fourwing saltbush, Atriplex canescens (Pursh) Nutt, a dominant and ecologically important shrub in southwestern USA rangelands. These fungi are characteristically identified as stained or pigmented hyphae and microsclerotia in the root cortex using conventional fungus staining methods. A. canescens roots colonized by DS fungi were stained with sudan IV and analyzed with differential interference microscopy (DIC). This method revealed substantial internal colonization of the cortex and vascular cylinder by vacuolated hyaline hyphae that were not evident when stained with trypan blue. Hyaline hyphae were internal extensions of melanized DS hyphae and microsclerotia. Sudan IV intensely stained lipids in fungal vacuoles, further enhancing visibility of internal hyaline hyphae. Melanized hyphae and microsclerotia were more abundant in roots sampled from dormant and relatively inactive plants while hyaline hyphae and lipid accumulation were most prevalent in roots of physiologically active plants. The polymorphic nature of DS fungal endophytes, their dynamic response to metabolic activity, and their similarities and differences relative to aseptate fungal colonization in A. canescens roots are discussed.