We studied the genetic structure of Mycosphaerella cryptica following natural infection of Eucalyptus globulus in a genetic trial. Results from this study indicated significant genetic variation within, and between, E. globulus families (three cloned and control-crossed F₂ and four open-pollinated families). Single-ascospore isolates were sampled from 72 E. globulus trees with contrasting levels of resistance within the trial, 21 E. nitens trees in an adjacent plantation, and five distant Eucalyptus plantation trees (two E. globulus 100–2400 km; two E. nitens 50–2200 km; one E. grandis × E. tereticornis hybrid 1500 km from the trial). Deoxyribonucleic acid from these isolates was scored for the presence/absence of 75 random amplified polymorphic DNA (RAPD) loci. Only 18 RAPD genotypes, which appeared to recombine rarely, were present among the 98 isolates, which indicates that M. cryptica is not strictly heterothallic. Cluster analysis using genetic distance revealed that M. cryptica genotypes from the field trial grouped into two clusters that matched differences in isolate culture morphology, indicating that the fungal population comprised two distinctive biotypes. The two biotypes differed markedly in their host interactions. Biotype 2 was found only on E. globulus, whereas biotype 1 infected both E. globulus and E. nitens eucalypt species. Within E. globulus, biotype 2 was almost exclusively collected on resistant trees whereas biotype 1 was found on both resistant and susceptible trees. The hypothesis that there could be specialisation of M. cryptica biotypes occurring at two levels, among host species and within host species, is discussed.