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<i>Ammannia arenaria</i> and <i>A. multifloras</i>, morphologically similar at the seedling stage, are the most common broad-leaved weeds in paddy fields. Our study showed that <i>A. arenaria</i> occupied more space than <i>A. multifloras</i> when competing with rice. However, <i>A. multifloras</i> germination has lower temperature adaptability. No difference in sensitivity to common herbicides between two <i>Ammannia</i> species was observed. Chloroplast (cp) genomes could be conducive to clarify their genetic relationship. The complete cp genome sequences of <i>A. arenaria</i> (158,401 bp) and <i>A. multiflora</i> (157,900 bp) were assembled for the first time. In <i>A. arenaria</i>, there were 91 simple sequence repeats, 115 long repeats, and 86 protein-encoding genes, one, sixteen, and thirty more than those in <i>A. multiflora</i>. Inverted repeats regions expansion and contraction and the phylogenetic tree based on cp genomes demonstrated the closely relationship between the two species. However, in <i>A. arenaria</i>, 20 single nucleotide polymorphisms in the CDS region were detected compared to <i>A. multiflora</i>, which can be used to distinguish the two species. Moreover, there was one unique gene, <i>infA</i>, only in <i>A. arenaria</i>. This study provides reliable molecular resources for future research focusing on the infrageneric taxa identification, phylogenetic resolution, population structure, and biodiversity of <i>Ammannia</i> species.