The widespread use of herbicides and antibiotics for the selection of transgenic plants has not been very successful with regards to commercialization and public acceptance. Hence, alternative selection systems should be established. In this study, we report the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional aadA, spectinomycin-based, selection system and the ipt selection system demonstrated that selection of transplastomic plants on media lacking cytokinin was as effective as selection on media containing spectinomycin. Proof of principle was demonstrated by transformation of the 3-ketoacyl-acyl-carrier protein synthase III (kasIII) gene into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system and were phenotypically normal despite IPT overexpression. Overexpression of KASIII resulted in a significant increase in 16:0 fatty acid levels and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates the generation of a novel positive plastid transformation system, which can be used, for the selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, typically not applicable to traditional based selection systems and could be used in combination with a negative selectable marker as part of a two step selection system to obtain homoplasmic plant lines.This article is protected by copyright. All rights reserved.