Duckweeds belong to the monocot subfamily of the Lemnoideae, which represents aquatic plants with great potential industrial applications. Here, we tested the power of next-generation sequencing to reconstruct the chloroplast genomes from three species in different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana from short reads of total plant DNA using the SOLiD platform. Because total DNA is so enriched by chloroplast DNA, we reasoned that the depth of reads would permit us to sequester the plastid reads computationally. Sequence reads were assembled into contiguous sequences (contigs) and scaffolds based on mate-paired reads using SOLiD software tools. To validate scaffolds, primers were selected to bridge gaps and confirm order of contigs experimentally. PCR products were also used to sequence gaps with the traditional ABI3730xl platform. Because 20% of overlapping information was obtained by two independent methods, we could assess the accuracy of the next generation sequencing platform. In addition, we could obtain the sequences of plastid genomes by the SOLiD platform from a single spot on a quadrant slide without purification. Although we found that the chloroplast genomes were conserved in gene number and organization in respect to the reference genome of L. minor, they exhibited higher nucleotide substitutions, abundant deletions and insertions in non-coding regions, exceeding the rates observed in species of the grasses. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.