The chloroplast represents an attractive compartment for light-driven biosynthesis of recombinant products, and advanced synthetic biology tools are available for engineering the chloroplast genome ( = plastome) of several algal and plant species. However, producing commercial lines will likely require several plastome manipulations, and this will present issues with respect to selectable markers: there are a limited number of markers available, these can be used only once in a serial engineering strategy, and it is undesirable to retain marker genes for antibiotic resistance in the final transplastome. To address these problems, we have designed a rapid iterative marker system for the green microalga Chlamydomonas reinhardtii that allows creation of marker-free transformants starting from wild-type strains. The system employs a dual marker encoding a fusion protein of E. coli aminoglycoside adenyltransferase (conferring spectinomycin resistance) and a variant of E. coli cytosine deaminase (conferring sensitivity to 5-fluorocytosine). Initial selection on spectinomycin allows stable transformants to be established and driven to homoplasmy. Subsequent selection on 5-fluorocytosine results in rapid loss of the dual marker through intramolecular recombination between the markers's 3'UTR and the 3'UTR of the introduced transgene(s). We demonstrate the versatility of the CpPosNeg system by serial introduction of reporter genes into the plastome. This article is protected by copyright. All rights reserved.