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Ceratophyllum is the only genus in the order Ceratophyllales and the family Ceratophyllaceae; however, the taxonomic classification of Ceratophyllum has long been debated. Previous studies have primarily relied on morphological characteristics and a few genetic markers for molecular analyses. However, inconsistencies between morphological characteristics and molecular data have often been observed, and previous studies have been insufficient in accurately establishing the phylogenetic placement of Ceratophyllum. Therefore, this study sequenced and assembled two Ceratophyllum (156,104–156,252 bp) by Illumina Truseq Nano sequencing. The overall chloroplast genome structure, GC content (38.2%), and gene content (113 genes) showed high conservation. This study identified four useful markers (trnK-rps16, trnL-ndhJ, ndhF-rpl32, and rpl32-trnL) that have not been previously examined in molecular research, which could be used as molecular markers. Phylogenetic analyses showed that Ceratophyllales is the sister group to eudicots. The genus Ceratophyllum taxa are a monophyletic group, and C. demersum is sister to C. demersum var. quadrispinum and C. demersum var. inerme. All chloroplast genes in Ceratophyllum exhibited nonsynonymous/ synonymous ratio well below one, indicating that they are evolving under purifying selection. This study provides chloroplast genome data for the Ceratophyllum genus, offering valuable molecular resources to support future taxonomic classification and phylogenetic studies.

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