We sequenced the plastid genome of four Medicago truncatula lines, Jemalong 2HA, Borung, Paraggio and R108-1, using the Illumina GAII platform. The barcoded, paired-end libraries were prepared from a pool of overlapping PCR fragments of the ~124 kb plastid genome. The sequence was assembled with a combination of de novo (Velvet) and reference-guided (BWA and CLC Genomics Workbench) assembly programs. Gaps were closed by Sanger sequencing PCR-amplified DNA fragments. Analysis of the data revealed significant intraspecies variation, the Jemalong 2HA ptDNA being the most similar to, and the R108-1 ptDNA being the most different from the reference A17 ptDNA. The new sequence information was useful to develop PCR-based plastid markers, which are being used to follow chloroplast inheritance.