Isolates of Gaeumannomyces graminis were sampled from 16 cereal crops throughout the cereal belt of southern Australia to determine the extent of genetic diversity and the scale of genetic differentiation among pathogen populations. Data from 13 isozyme and 4 RFLP loci differentiated 79 multilocus genotypes among the 320 isolates analysed. All 17 loci differed significantly in allele frequencies across all populations and significant levels of genetic differentiation were detected between most populations. Genetic differentiation among host groups was high (GST = 0.307) and groups of populations from barley, oats and wheat were significantly different. The average genetic identities among populations were low and populations formed genetically related groups based on similarities in recent cereal cropping histories and not geographical origins. Collectively, these analyses indicate restrictions to inter-population gene flow within G. graminis and imply that population differentiation results from genetic drift and host-mediated selection by different cereal species.