AbstractBackgroundOrder Chaetophorales currently includes six families, namely Schizomeridaceae, Aphanochaetaceae, Barrancaceae, Uronemataceae, Fritschiellaceae, and Chaetophoraceae. The phylogenetic relationships of Chaetophorales have been inferred primarily based on short and less informative rDNA sequences. This study aimed to phylogenetically reconstruct order Chaetophorales and determine the taxonomic scheme, and to further understand the evolution of order Chaetophorales.ResultsIn the present study, seven complete and five fragmentary chloroplast genomes were harvested. Phylogenomic and comparative genomic analysis were performed to determine the taxonomic scheme within Chaetophorales. Consequently, Oedogoniales was found to be a sister to a clade linking Chaetophorales and Chaetopeltidales. Schizomeriaceae, and Aphanochaetaceae clustered into a well-resolved basal clade in Chaetophorales, inconsistent with the results of phylogenetic analysis based on rDNA sequences. Comparative genomic analyses revealed that the chloroplast genomes of Schizomeriaceae and Aphanochaetaceae were highly conserved and homologous, highlighting the closest relationship in this order. Germination types of zoospores precisely correlated with the phylogenetic relationships.Conclusionschloroplast genome structure analyses, synteny analyses, and zoospore germination analyses were concurrent with phylogenetic analyses based on the chloroplast genome, and all of them robustly determined the unique taxonomic scheme of Chaetophorales and the relationships of Oedogoniales, Chaetophorales, and Chaetopeltidales.