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Arbuscular mycorrhiza (AM) fungi form symbiotic relationships with the majority of land plants and are known for their positive effects on plant P acquisition and soil quality. The extramatrical growth of the mycelium is a key factor in nutrient acquisition by the symbiont. Soil grinding and extraction/fractionation of lipids were used in a field experiment to identify probable sources of the AM biomarker C16:1cis11 and its functional significance during reproductive growth of corn (Zea mays L.). Chambers, enclosed with a 1mm mesh fabric to allow roots and hyphae to pass into the enclosed soil volume, were installed in two field sites cropped to continuous corn in central Nebraska. The chambers were installed at tasselling and removed after 3, 6 and 9 weeks. Soil from the chambers was analyzed by ester-linked fatty acid (EL-FAME) and chloroform–methanol fatty acid (CM-FAME) analysis. We also separated and analyzed the neutral lipid (NLFA), glycolipid (GLFA) and phospholipid (PLFA) fatty acid fractions. Roller milling the soil gave up to two-fold increases in the recovery of EL- and CM-FAMEs common to saprophytic fungi (C16:0, C18:1cis9, C18:2cis9,12) and AM fungi (C16:0, C16:1cis11, C18:1cis11) but not those specific to bacteria or fauna. Resistant AM fungal structures were enriched in NLFA and GLFA C16:1cis11, but not PLFA, indicating that storage lipids and possibly cell-wall lipids are released by roller milling. Similar proportional increases in C16:1cis11 on roller milling indicates that mild alkaline hydrolysis (EL-FAME) is as inefficient as chloroform–methanol (CM-FAME) in extracting lipids from AM spores. EL- and CM-FAME C16:1cis11 increased by one-third between R5 and R6, indicating C allocation from the plant to the AM fungus during the reproductive stages of corn. This increase was attributed to accumulation of NLFA and GLFA in lipid-containing structures of the extramatrical mycelium and AM structures within roots, not increased sporulation. We propose EL-FAME C16:1cis11 as a simple measure of AM biomass in soils that largely reflects the AM hyphal network important to nutrient acquisition by the plant.