Monoic, dioic, and hermaphrodite flower types complicate spinach breeding and cultivar development. The availability of haploid plants will definitely accelerate spinach breeding; however, there are currently no reports in the literature about the use of tissue culture techniques to obtain spinach haploids. Therefore, in this study, pollination with irradiated pollen and anther culture methods were used to obtain haploid plants in spinach. For anther culture, three spinach varieties (Koto F1, Favorit F1, and Greenstar F1) and four different nutrient media were tested to obtain haploid embryos. Significant outcomes were not achieved from anther culture, and only two plants were obtained from all the experiments. Gynogenesis studies using irradiated pollen were performed with the same three spinach varieties and six gamma ray doses (100, 150, 200, 300, 400, and 500 Gy) from (cobalt60) Co60. Murashige and Skoog (MS) nutrient medium containing 1 mg·L-1 indoleacetic acid (IAA) was used for embryo germination. Current findings revealed that the varieties produced responded differently to the various doses of radiation. A total of 3414 embryos and 1710 plants were obtained from experiments carried out for 2 years. Considering the numbers of embryos and plants per 100 seeds, the Favorit F1 variety provided better results than the other two varieties. However, significantly different outcomes were not achieved with regard to irradiation doses. Embryos were observed at all doses tested. Flow cytometry analyses that were carried out on regenerated plants revealed whether the plants were diploid or doubled haploid, and molecular analyses revealed that diploids resulted from spontaneous chromosome doubling. The current findings offer significant results for spinach breeding using haploids.