To enable quantification of mycelial abundance in mixed-species environments, eight new TaqMan® real-time PCR assays were developed for five arbuscular mycorrhizal fungal (AMF, Glomeromycota) taxa. The assays targeted genes encoding 18S rRNA or actin, and were tested on DNA from cloned gene fragments, from spores, mycelia, and from root-free soil, and on reverse-transcribed rRNA templates from entire mycelia and from colonized roots. The assays showed high specificity, sensitivity, and reproducibility, enabling reliable quantitation over broad ranges of template molecules. From cultured mycelia, DNA and RNA measures both correlated with spore number rather than extraradical hyphal length, and epifluorescence microscopy identified pronounced heterogeneity in vitality and nuclear distribution in hyphae. Root colonization was also spatially heterogeneous, as shown by a mixing experiment with root fragments of different length. Therefore, although real-time PCR can reproducibly and accurately quantify AMF nucleic acids, these are poorly correlated with visual measures because of spatial heterogeneity.