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Chilli (Capsicum annuum), which belongs to the family Solanaceae, is an important spice crop cultivated in tropical and subtropical countries. A Protocol for chili plant regeneration with hypocotyl and cotyledon explants was established. The study was conducted to observe the effect of genotypes, culture conditions and growth regulators on plant regeneration of chili pepper genotypes (Seedex Pepper (SP), Loungi, Tatapuri, and Sanam) grown in Pakistan including. For both hypocotyl and cotyledon explants, SP was found to be the most sensitive among the tested genotypes tested. Maximum mean callus induction rate of hypocotyl was 76.6% in SP followed by TP (73.3%); Sanam (57.3%); Loungi (43.3%) respectively. Whereas in case of cotyledon maximum inductions rate was found in Loungi (62.0%) followed by SP (60); Sanam (57.3); TP (46) individually. According to the observations maximum plant regeneration was found in genotype SP (9%), followed by Loungi (1.8%); Sanam (1.5%) but no plant regeneration was found in TP. A. tumefaciens LBA4404 with the 35S GFP/pFGC construct were used to evaluate hypocotyl and cotyledon explants for transformation. The effects of co-cultivation at different temperatures (22 and 25oC), photoperiods (16 hours of light, 8 hours of darkness, and full darkness), and co-cultivation periods were studied. GFP assays revealed that the putative transgenic calli were not transformed and died after 40-60 days. The experiment was repeated ten times to see the success rate of the regeneration system. In this study a protocol for chili plant regeneration was developed for callus induction, plant regeneration, and plant transformation. Because developing a viable chilli tissue culture and plant regeneration system is difficult, this study was designed to help develop one. SP was found to be the most suitable among the four genotypes used for regeneration. [ABSTRACT FROM AUTHOR]