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Myriophyllum , among the most species-rich genera of aquatic angiosperms with ca. 68 species, is an extensively distributed hydrophyte lineage in the cosmopolitan family Haloragaceae. The chloroplast (cp) genome is useful in the study of genetic evolution, phylogenetic analysis, and molecular dating of controversial taxa. Here, we sequenced and assembled the whole chloroplast genome of Myriophyllum spicatum L. and compared it to other species in the order Saxifragales. The complete chloroplast genome sequence of M. spicatum is 158,858 bp long and displays a quadripartite structure with two inverted repeats (IR) separating the large single copy (LSC) region from the small single copy (SSC) region. Based on sequence identification and the phylogenetic analysis, a 4-kb phylogenetically informative inversion between trn E- trn C in Myriophyllum was determined, and we have placed this inversion on a lineage specific to Myriophyllum and its close relatives. The divergence time estimation suggested that the trn E- trn C inversion possibly occurred between the upper Cretaceous (72.54 MYA) and middle Eocene (47.28 MYA) before the divergence of Myriophyllum from its most recent common ancestor. The unique 4-kb inversion might be caused by an occurrence of nonrandom recombination associated with climate changes around the K-Pg boundary, making it interesting for future evolutionary investigations., Competing Interests: The authors declare no conflict interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)