Mucilage is a class of polysaccharides found in some plants that have pharmaceutical effects as anti-hemorrhoids. Alyssum is one such species; its seeds produce mucilage that possesses pharmaceutical properties. The aim of this study was to optimize the conditions for callus production and mucilage synthesis in Alyssum species in a tissue culture procedure. In the study presented here, callus initiation in different genotypes of Alyssum species (A. inflatum, A. lepidium, and A. strigosum) has been investigated for the first time. Different combinations of 2,4 Dichlorophenoxy acetic acid (2,4 D), Kinetin (Kin) and Benzyl amino purin (BAP) were used to optimize callus initiation frequency and callus growth rates (CGR) in hypocotyl explants. The highest rates of callus induction (%) and callus growth rates (CGR) were achieved with 2.5 mg • 1^-1 2,4D + 0.1 mg • 1^-1 Kin and 3 mg • 1^-1 2,4D + 0.1 mg • 1^-1 Kin, respectively. The highest callus induction was recorded for A. inflatum. The mucilage content was estimated in callus cultures of different explants. The novel finding of the study is the superiority of the mucilage extracted from the callus rather than the seed, which is about ten times higher. The highest mucilage production (g/g dry weight) was obtained with Murashige and Skoog (MS) medium supplemented with 2 mg • 1^-1 2,4 D and 0.1 mg • 1^-1 Kin. Among the genotypes investigated, Kerman (A. lepidium) was found to produce the most mucilage (0.51 g/g d.w.) in its callus. The proposed method is beneficial for mucilage production from Alyssum sp. using in vitro cell culture.