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Conserved chloroplast (cp) DNA primer pairs are useful in plant molecular genetics, evolution and ecology. We have designed 20 conserved cpDNA primer pairs that, in combination with 18 previously described ones, amplify overlapping fragments (mean size of 2.5 kb) spanning the large single copy (LSC) region from Eudicots. These 38 primer pairs as well as eight primer pairs flanking cpDNA microsatellites were tested on 20 plant species belonging to 13 families. At least 79% and up to 100% of the LSC (> 86 kb) can be amplified. Many primer pairs are robust and work with all species.