The effects of the genotypes of donor plants and three steps of culture media (induction, differentiation, and regeneration media) were evaluated in unpollinated ovary culture of cucumber (Cucumis sativus L.) to obtain the best combination of culture media for doubled haploid (DH) production of recalcitrant cultivars. Embryo-like structure (ELS) formation was significantly affected by differentiation and regeneration media. The highest percentages of ELS formation (59.89%) were obtained when the ovaries were cultured on the D2 second step culture medium and transferred to the newly developed MS medium supplemented with several plant growth regulators and additives, MST3 , the third step culture medium. However, there was no significant effect of the genotypes and induction (first step culture) media on ELS and callus formation. The ELSs developed into shoots when transferred to the third step culture media and complete plantlets were obtained after rooting on the MS medium with no supplement (MS0). Among the 10 regenerated plantlets that survived after transplanting to soil, three were identified as haploid (H) plantlets (2n = x = 7), one was a triploid plantlet (2n = 3x = 21), and six were diploid plantlets (2n = 2x = 14). Inter-simple sequence repeat analysis confirmed that these six diploids were DH plantlets. These results suggest that the newly developed media are useful for the future production of cucumber DHs.