The commercial use of onion hybrids is preferred by producers. In contrast, the production of hybrid onion seeds is extremely inefficient. This is due to the use of lineages obtained by successive self-fertilizations assuming the effect of inbreeding depression per se. Therefore, it is necessary to understand new alternatives to reduce the effect of inbreeding depression in the strains. The objective of this study was to evaluate possible differences in polymorphism and levels of heterozygosity of strains obtained from intercrossed and self-fertilized plants. Twelve onion populations belonging to Bayer’s breeding program (Granex, IPA-1, IPA-2 and IPA-3) were used, obtained by self-fertilization of one plant, intercrossing of two plants or intercrossing of three plants. Three individuals from each strain were used in the analyzes. The amplifications were performed using 8 microsatellite primers with greater polymorphism, according to germplasm characterization studies carried out by CITA. Heterozygosity generally decreases with self-fertilization and increases as more plants are used in the cross. The SSR markers used in the present study were efficient in detecting variability in different genetic backgrounds. With the results obtained, it is suggested to carry out the obtaining of hybrids between the different combinations and to analyze the performance per se of the different modalities of obtaining strains proposed in the present research.