Acrocomia aculeata (Arecaceae), a palm endemic to South and Central America, is a potential oil crop. Knowledge of the mating system of this species is limited to its reproductive biology and to studies using molecular markers. The present study analyzed genetic diversity between its developmental stages and determined its prevailing mating system in order to support genetic conservation and breeding programs. We tested nine microsatellite markers in 27 mother trees (adult plants) and 157 offspring (juvenile plants) from the southeastern region of Brazil. Heterozygosity levels differed between the two studied life stages, as indicated by the fixation index of adult and juvenile trees, suggesting that selection against homozygotes occurs during the plant life cycle. The mating system parameters analyzed indicate that A. aculeata is predominantly outcrossing (allogamous). However, its low levels of selfing suggest that there is individual variation with regard to self-incompatibility, which can be a survival strategy in isolated or fragmented habitats. Deviations in variance effective size were detected because of high mating rates among relatives and correlated matings. These findings indicate that the main source of inbreeding results from biparental inbreeding in the population and that the progenies are predominantly composed of full-sibs. The information provided by this study on the ecology and reproduction dynamics of A. aculeata should be useful to both breeding and genetic conservation programs, allowing the development of more precise mathematical models and the estimation of the appropriate number of mother trees for seed collection.