A laboratory study was conducted to establish a broadly applicable in vitro regeneration and transformation methods for increasing β-carotene content in pepper fruits. Therefore, two different pepper genotypes were analyzed with regard to their efficiency for regeneration and genetic transformation in vitro. Cotyledons were used in vitro from young seedlings as target tissue for regeneration and transformation of pepper. Over expression of β-Lcy gene via Agrobacterium-mediated transformation was used. The presence of the transgene was assayed on leaf DNA via PCR and biochemical analysis using HPLC. Results strongly showed that there were significant differences between pepper genotypes. The transgenic pepper plants showed a significant increase in fruit β-carotene content reaching 7 to 10 folds according to the genotype. The fruits from these plants displayed different colour phenotypes, from orange to orange-red. The difference in the fruit colour phenotypes reflects the β-carotene ratio, which ranged from 4.5 mg/100g DW in the wild plants to 45.0 mg/100g DW in the transgenic plants of genotype Balady during maturity stage. Concerning to the genotype Topepo Rosso, the β-carotene content was increased from 5.2 mg/100g DW in the wild plants to 36.39 mg/100g dw in the transgenic plants during maturity stage.