An in vitro plant generation and genetic transformation protocol was established in sesame (Sesamum indicum L. cv Rama) through biolistic particle gun bombardment. 5-day-old apical, meristematic tissues of in vitro-germinating seedlings were used as explants. 10–15 Multiple shoots were generated from each explant using Murashige and Skoog basal medium containing 18.0 µM benzylamino purine and 5.37 µM naphthalene acetic acid. Four independent sets of transformation were carried out and each set consisted of three independent experiments each comprising three replications with 30 explants per replication. A synthetically designed bialaphos resistance gene (bar) was used for transformation. The positive transformants containing the bar gene were selected in growth medium containing 2.5 mg/L bialaphos. Green shoots recovered from bombarded explants were subjected to root development on Murashige and Skoog basal medium containing 5.37 µM naphthalene acetic acid. The rooted shoots were established in soil and grown to maturity in greenhouse. Polymerase chain reaction (PCR), Southern and reverse-transcription PCR, real-time quantitative PCR, western blot and enzymatic assay of four putative transformants from independent sets provided evidence for full-length gene integration as well as high level expression of the transgene. Analysis of the T1 plants revealed a stable inheritance of the transgene through the progenies. This is the first report of biolistic mediated stable transformation of sesame and should pave the way for future genetic engineering strategies to be employed for improvement of this very important oil-seed crop.