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The aquatic perennial herb Sagittaria trifolia L. commonly known as arrowhead, has been utilized in China both as a culinary vegetable and in traditional medicines. Characterizing the phylogenetic relationships and genetic diversity of arrowheads is crucial for improved management, conservation, and efficient utilization of the germplasm resources associated with this species. Herein, we presented the phenotypic traits and genome-wide DNA marker-based analyses of 111 arrowhead accessions, most of which were from China. Cluster analysis revealed that arrowhead could be categorized into two clusters based on 11 phenotypic traits, with Cluster 1 comprising two subclusters. All accessions were clustered into three sub-clusters based primarily on leaf shape and tuber weight. A set of 759,237 high-quality single-nucleotide polymorphisms was identified and used to assess the phylogenetic relationships. Population structure and phylogenetic tree analyses suggested that the accessions could be classified into two major groups, Group I was further subdivided into two subgroups, aligning with the clusters identified through morphological classification. By employing Sagittaria lichuanensis as an outgroup, the rooted tree revealed that the evolutionary relationships within the three groups followed a progression from Group I-1 to Group I-2 and finally to Group II. The landraces were clustered into one group along with the remaining wild accessions. The level of genetic diversity for Group I (π = 0.26) was slightly lower than that which was estimated for Group II (π = 0.29). The lowest pairwise differentiation levels (Fst, 0.008) were obtained from the comparison between groups I-2 and II, indicating that the two groups were the most closely related. This study provides novel insights into germplasm classification, evolutionary relationships, genomics and arrowhead breeding. [ABSTRACT FROM AUTHOR]