Arbuscular mycorrhizal (AM) fungi (Glomeromycota) are thought to be the oldest group of asexual multicellular organisms. They colonize the roots of most land plants, where they facilitate mineral uptake from the soil in exchange for plant-assimilated carbon. Cells of AM fungi contain hundreds of nuclei. Unusual polymorphism of ribosomal DNA observed in individual spores of AM fungi inspired a hypothesis that heterokaryosis—that is, the coexistence of many dissimilar nuclei in cells—occurs throughout the AM fungal life history. Here we report a genetic approach to test the hypothesis of heterokaryosis in AM fungi. Our study of the transmission of polymorphic genetic markers in natural isolates of Glomus etunicatum, coupled with direct amplification of rDNA from microdissected nuclei by polymerase chain reaction, supports the alternative hypothesis of homokaryosis, in which nuclei populating AM fungal individuals are genetically uniform. Intrasporal rDNA polymorphism contained in each nucleus signals a relaxation of concerted evolution, a recombination-driven process that is responsible for homogenizing rDNA repeats. Polyploid organization of glomeromycotan genomes could accommodate intranuclear rDNA polymorphism and buffer these apparently asexual organisms against the effects of accumulating mutations.