Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.