This is the first report of successful regeneration of haploid lines in iris (Iris pseudacorus) developed by in situ parthenogenesisfollowed by embryo rescue. Native genotypes of I. pseudacorus (2n = 38) were prepared as female parents and I. spuria was set as the males.Anthers collected from the pollen donor were irradiated by X-ray with doses of 0, 100, 200, 300, and 400 Gy in 2012. A second roundof experiments was conducted in 2013 with 300 and 400 Gy. The ovaries from each pollinated flower were collected and cultured andthe produced plantlets were then acclimatized. Chromosome counting and flow cytometry were used to determine the ploidy levels. Theviability of irradiated pollens was also evaluated. Pollination with irradiated pollen at doses ranging from 100 to 400 Gy can significantlyreduce fruit set, particularly at higher doses (300 and 400 Gy). The embryo production was reduced in I. pseudacorus as doses increased;the highest doses led to reduction in the number of regenerants with the formation of just 6 and 4 embryos at 300 and 400 Gy, respectively.Haploid plantlets were produced when the pollen grains were exposed to 300 and 400 Gy of X-ray. Irradiation at 100 and 200 Gy was foundto be insufficient to cause pollen sterility. The best dose for haploid production in I. pseudacorus is hereby found to be 300 or 400 Gy of X-ray.