Gynogenesis of four onion cultivars was induced, starting from ovules and ovaries, using a 2-step culture procedure. The induction frequency was much higher from ovaries and was strongly affected by genotype. Thidiazuron (2 mg/l) had a positive effect on regeneration and subsequent in vitro culture of regenerants. Esterase isozyme analysis showed that 59% of regenerants were homozygous. Chromosome counting of root tip cells and flow cytometry of leaf tissue showed haploid, doubled haploid and mixoploid structures. Within cloned plants, some showed segregation of the ploidy level with a tendency towards endoreduplication. Analysis of random amplified polymorphic DNA (RAPD) profiles of homozygous regenerants revealed several missing bands compared with donor plants, but micropropagated clones of different ploidy level were identical among each other. Gametoclonal variation was detected by the appearance of a novel RAPD band, present in two out of 12 regenerants and not in the donor plants.