Allium hirtifolium (Amaryllidaceae), called “Mooseer” in Iran, is a bulbous plant with high medicinal values. It is native and endemic to the North West to central and South West of Iran. In the present study, regeneration plants with chromosomal changes are produced through in vitro mature and immature endosperm culture. In our study, both cultured endosperms formed yellow- green calli induced in the MS medium supplemented with (1.5 or 2 mg·L-1) 2,4-dichlorophenoxyacetic acid (2,4- D), (0.5 or 1 mg·L-1) α-naphthalene acetic acid (NAA) plus (0.5 or 1 mg·L-1) 6-furfurylamino purine (Kin) and 6-benyzlaminopurine (BAP). The highest callus induction in mature (87.62) and immature (62.49) endosperms was achieved on a medium with 1 mg·L-1 NAA and 1 mg·L-1 BAP. Regenerated shoots were transferred for bulblet growth to investigate their potential for bulblet production. Regeneration of plantlets from endosperm tissue shows high chromosome variations and polyploidy. Flow cytometry analysis and chromosome observation confirmed that diploid (2n= 2x= 16), triploid (2n= 3x= 24), tetraploid (2n= 4x= 32) and aneuploid plantlets were achieved in these regenerated plants. The comparison of stomatal density and size in complete plantlets has revealed that with the increase in ploidy level, stomatal density decreased because of enlargement in stomatal length.