AbstractA survey of electrophoretically detectable genetic variation in the only known population of Nothomyrmecia macrops Clark (from N. W. Eyre Peninsula, South Australia) revealed one polymorphic locus (amylase) among 16 studied, and a mean heterozygosity (Ĥ) of 0.032, a value lower than those reported for most other insects, but consistent with previous comparable estimates for ants and other Hymenoptera. Variability at the amylase locus is considerable, with 4 alleles present at frequencies > 0.05. There is no evidence of inbreeding, genotype frequencies being in Hardy‐Weinberg equilibrium. An examination of genotype frequencies within 4 nests demonstrates that workers from the same nest are not always sibs, and a regression analysis of relatedness among worker nestmates yields a mean estimated coefficient of relatedness (b) of 0.251, based on 2 different diallelic combinations. The same analysis applied to samples of workers taken foraging on separate, single Eucalyptus trees gives a mean b of only 0.084 (based on three diallelic combinations), a result consistent with field observations of an overlap of foraging areas (and probably low territoriality) between nests. The relatedness estimates must be interpreted with caution because of limited sample sizes. Additional field studies and electrophoretic analyses are required to assess the possible contributions to the observed patterns of relatedness from polygyny, multiple insemination of the queen(s), and worker interchange between colonies.