Four maize hybrids (122, 168, 173 and pioneer) were investigated to determine their genotypic effects on callogenesis response to 2,4-D (2,4 dichlorophynoxyacetic acid) and in vitro salt tolerance using NaCl. A callus was initiated from mature embryos cultured on three MS media supplemented with 0, 2 and 4mg/l of 2,4-D where the level 0 is the control. The produced calli from the medium which contains 2 mg/L 2,4-D for each genotype were then exposed to four  salinity levels (i.e. 0, 1500, 3000 and 6000 ppm) of NaCl, respectively to evaluate some physiological aspects of the produced calli from the genotypes. The results of ANOVA revealed that the mean squares of genotypes at all levels of 2,4-D were significantly indicated the presence of differences between them for all in vitro traits. Furthermore, the mean squares of levels and genotype × levels interaction were highly significant for all traits. This indicated that these genotypes gave different responses at different 2,4-D levels. The genetic variation was high and positive with respect to the in vitro traits at each 2,4-D level. This indicated that these traits are mainly controlled by genetic factors. This finding was emphasized by the heritability values, which were more than 80% for all studied traits at each 2,4-D level. In relation to callus response to salt stress, the results indicated that the presence of significant differences between these genotypes for ion content in callus cells (K⁺%, Na⁺%, Na⁺/K⁺ and Cl^-%) with respect to the four salinity levels except for K⁺% at S₁,S₃ levels, Na⁺% at levels S₁, S₂, S₃, Na⁺/k⁺ at levels S₀, S₁, S₃ and Cl^-% at levels S₂, S₃.  Regarding to, the organic solutes accumulation (sugar and proline mg/g f.wt), the magnitudes of the mean squares for genotypes were significant at four salinity levels except for proline mg/g f.wt at S₃ level. For membrane permeability, relative growth rate and water content, the magnitudes of the mean squares for genotypes were significant at four salinity levels except for water content at levels S₀, S_3.The results revealed that the genetic variation was high and positive for ion content in callus cells (K⁺%, Na⁺%, Na⁺/K⁺ and Cl^-%), organic solutes accumulation (sugars and proline), membrane permeability and relative growth rate as well as water content with respect to the four levels of salinity. This finding is emphasized by the heritability values, which were more than 80% for most of studied traits.