Plant tissue culture technique was used to develop a regeneration procedure in local Pakistani cotton varieties. Three cotton varieties Coker 312, CEMB66 and Klean cotton were cultured on MS media supplemented with vitamins and growth regulators. The results showed that the regeneration potential of Coker 312 was higher, CEMB 66 moderate while Klean cotton lacks the cotton regeneration potential. The molecular analysis identified the Somatic Embryogenesis Receptor Kinase3 (SERK3) gene and its expression analysis confirmed that it plays role for embryo formation through tissue culture. Its low expression resulted in calli to die rather than forming embryos. Therefore, SERK3 gene is considered important for callus induction and its development into primary embryogenic and embryogenic stage. Further investigations will be helpful to understand the mechanism of regeneration potential of cotton through tissue culture.