Anther culture is a biotechnology technique that can be used for the production of pure lines. The aims of this investigation were to induce embryogenic callus from major and minor culms of Thai aromatic rice cultivars and to subsequently regenerate double-haploid green plantlets by the application of exogenous polyamines. Embryogenic callus derived from anther culture was successfully induced in varieties KDML105, Homjan (HJ), and Pathumthani 1 (PT1). Production of embryogenic callus from anthers collected from the major culms was greater than those collected from the minor culms, especially in cultivar HJ. Plantlet regeneration in the three rice cultivars was observed from embryogenic callus and was highest, at 12.1%, from variety HJ treated with 0.5 mM spermidine. Plantlet regeneration from anther-derived embryogenic callus was dependent on the plant genotype, the types of exogenous polyamines, and the interactions of these factors. The percentage of haploid plantlets regenerated in PT1, KDML105, and HJ were 68.1%, 70.7%, and 78.5%, respectively. Only haploid plantlets were treated with colchicine for double-haploid production. This investigation has increased the knowledge of both embryogenic callus induction and plantlet regeneration in aromatic rice and has lead to the development of a pure, double-haploid line for the use in rice breeding programs in Thailand.