Nicotiana tabacum is a model widely used in functional genomics with transgenesis; however, genetically modified organisms are not accepted by consumers in Europe. Targeted mutagenesis as a non-transgenic approach was assessed on a demonstration gene involved in alkaloid metabolism. A population of 4.000 EMS-mutagenized M2 families was created. Single Strand Conformation Polymorphism (SSCP) was used to target mutations. Eleven putative mutants were identified by screening 1344 M2 families. Mutations identified in DNA pools were validated by sequencing. Individual plants carrying missense or truncation mutations were studied for their phenotype. Homozygous plants for one truncation were identified, and the expected phenotype was observed in the field. These plants have been used as genitors to introduce this mutation into elite lines. Backcrosses are being performed to recover the elite line background in combination with SSCP analysis to track the mutation. The amphidiploid nature of tobacco avoids problems related to fertility. The efficiency of this method to create novel genetic variation and to develop cultivars has been demonstrated for the first time in tobacco.