For the first time, report the regeneration of haploid line in Iris (I. pseudacorus) developed by in situ parthenogenesis followed by embryo rescue. Also the viability of irradiated pollens has been evaluated. Native genotypes of I. pseudacorus (2n = 38) as female and I. spuria as male parents were prepared. Anthers collected from the pollen donor were irradiated by X-ray with 0, 100, 200, 300 and 400 Gy in 2012 and with 300 and 400 Gy in 2013. The ovaries from each pollinated flower were collected, cultured and the produced plantlets were acclimatized. Chromosome counting and flow cytometery was used for determination of the ploidy level. Pollinated with irradiated pollen at doses ranging from 100 to 400 Gy significantly reduce seed production, in particular at higher doses (300 and 400 Gy). With increasing doses the embryo production reduced in iris, the highest doses embryo production reduced 6 and 4 embryo in 300 and 400 Gy respectively. Haploid plantlets were produced when 300 and 400 Gy of X-ray. Pollens irradiated at 100 and 200 Gy doses are insufficient for pollen sterility. The best dose for haploid production in Iris is 300 or 400 Gy X-ray.