A laboratory study was conducted to develop a broadly applicable in vitro regeneration method for tomato. Therefore, ten different tomato genotypes were analyzed with regard to their efficiency for regeneration in vitro. Cotyledons were used in vitro from young seedlings as target tissue for regeneration of tomato. Healthy multiple shoot regeneration was obtained from explants of cotyledon cultured on MS medium containing 2.0 mg/l BAP and 0.1 mg/l IAA. The maximum root induction from the regenerated shoots was achieved on half the strength of MS medium supplemented with 0.1 mg/l NAA. Regenerated plants appeared morphologically normal and set flowers and fruits with seeds that could germinated normally. Results strongly showed that there were significant differences between tomato genotypes. Generally the highest values of all regeneration parameters were obtained from genotypes Pantano Romanesco and Strain B. Genotype Ponderosa Seligocia had the lowest regeneration parameters while other genotypes were moderate response. The nutritional and antioxidant composition of regenerated ten tomato genotypes at full ripe stage was determined. The analysed components included ascorbic acid, phenolic compounds, total sugar, lycopene and β-carotene contents. Results indicated that the potential efficiency of biochemical compounds vary considerably with genotype. Genotype Strain B had the maximum content of ascorbic acid (19.3 mg/100 g fresh weight). Genotype Red Stone had the highest phenolic content of 34.4 mg/100 g fw at their full red ripe stage. The highest tomato genotype in present investigation of total sugar content was Impreal (4.5%). Petomech genotype was a promising genotype in terms of lycopene content (13.3 mg/100 g fw), while the yellow tomato genotype Pomodoro Banana Giallo had the highest content of β-carotene (4.6 mg/100 g fw). Overall, these tomato genotypes could contribute as sources of important nutritional and antioxidants related to the prevention of chronic diseases associated to oxidative stress, such as cancer and coronary artery disease.