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The order Nymphaeales, consisting of three families with a record of eight genera, has gained significant interest from botanists, probably due to its position as a basal angiosperm. The phylogenetic relationships within the order have been well studied; however, a few controversial nodes still remain in the Nymphaeaceae. The position of the Nuphar genus and the monophyly of the Nymphaeaceae family remain uncertain. This study adds to the increasing number of the completely sequenced plastid genomes of the Nymphaeales and applies a large chloroplast gene data set in reconstructing the intergeneric relationships within the Nymphaeaceae. Five complete chloroplast genomes were newly generated, including a first for the monotypic Euryale genus. Using a set of 66 protein-coding genes from the chloroplast genomes of 17 taxa, the phylogenetic position of Nuphar was determined and a monophyletic Nymphaeaceae family was obtained with convincing statistical support from both partitioned and unpartitioned data schemes. Although genomic comparative analyses revealed a high degree of synteny among the chloroplast genomes of the ancient angiosperms, key minor variations were evident, particularly in the contraction/expansion of the inverted-repeat regions and in RNA-editing events. Genome structure, and gene content and arrangement were highly conserved among the chloroplast genomes. The intergeneric relationships defined in this study are congruent with those inferred using morphological data. [ABSTRACT FROM AUTHOR]