Osmotic stress resulting from cell dehydration is caused by water deficit, and leads to the disruption of many cellular functions. The effect of osmotic stress was assessed on seedling and callus parameters of six grass pea (Lathyrus sativus) genotypes under in vitro conditions. Osmotic stress was induced in the agar media by adding sucrose concentrations corresponding to –4.5 and –8 bar. The growth and morphological traits of in vitro seedlings and calli were evaluated 12 and 30 days after exposure to osmotic stress, respectively. In seedlings, genotypes differed significantly for most traits, while the osmotic potential significantly influenced on the dry weight of the rootlet, as well as the fresh weight of the plumule and seedling, and the dry weight of plumule. Rootlet, plumule, and seedling dry matter decreased under osmotic stress. In turn, the higher dose of sucrose led to a reduction in seedling growth of the genotypes. In callus, the genotypes varied significantly for callus fresh and dry weights, as well as final callus size. The effect of osmotic potential was significant on callus fresh weight and relative water contents of the calli. Based on the tolerance indices, ICARDA-I can be recommended as a osmotic stress-tolerant genotype.