Genetic diversity of natural populations of the two species Naegleria gruberi and N. australiensis collected at the same time in the same area was studied using multilocus enzyme electrophoresis (MLEE analysis). Reference strains and related species were also analysed for comparison. Five loci were found to be polymorphic and allowed identification of 19 different multilocus genotypes among 67 isolates – eight for N. gruberi and 11 for N. australiensis – none being common to the two species. Large departures from Hardy–Weinberg equilibrium were found at all the loci, with significant heterozygote deficiencies in most cases. The two species exhibited strong linkage disequilibrium and the predominance of a few multilocus genotypes within each species was observed. These lines of evidence strongly suggest non-random association of alleles, leading to the conclusion that N. gruberi and N. australiensis have predominantly clonal genetic structures in the wild. Populations consist of a few major clones with some closely related ones – a structure found for several parasitic protozoans and bacteria. This contrasts with the panmictic structure found for N. lovaniensis, another species of this genus, and suggests that closely related species within the genus have evolved to have different reproductive strategies.