To study the genetic variability in Glomus mosseae and Gigaspora margarita sequence similarities of ITS regions of rDNA were analysed from spores collected from a permanent pasture and from pot cultures. PCR amplification with the primers ITS1 and ITS4 was performed and products were cloned and sequenced. The sequences from single spores of G. mosseae and Gi. margarita confirmed that there is variation in the ITS region in a single spore. Phenetic analysis of sequences from both species supported the morphological identification which placed the species into two separate groups. Through the analysis of these sequences an estimate of genetic diversity was derived which clearly showed that the three field spores of G. mosseae were at least 2–5 times more genetically diverse than one single spore (field) and a pool of spores (pot culture) of Gigaspora margarita. This demonstrates that a high degree of variation exists in this natural population of G. mosseae.