Pseudolysimachion pyrethrinum var. gasanensis (Gasan spike speedwell) is a valuable Korean endemic variety with significant horticultural potential. Despite its morphological distinctiveness, its taxonomic status and evolutionary position have remained a subject of debate. In this study, we assembled and characterized the first complete chloroplast (cp) genome of P. pyrethrinum var. gasanensis using high-throughput sequencing. The complete plastome is 152,251 bp in length, exhibiting a typical quadripartite structure with a large single-copy (LSC) region (83,191 bp), a small single-copy (SSC) region (17,690 bp), and two inverted repeats (IRs) (25,685 bp each). The genome contains 133 genes, including 88 protein-coding, 37 tRNA, and 8 rRNA genes. Genomic analysis identified 42 simple sequence repeat (SSR) units across 38 distinct loci, predominantly mononucleotide A/T motifs, which serve as potential molecular markers for variety-level identification. Selective pressure analysis revealed that the majority of protein-coding genes are under strong purifying selection (Ka/Ks < 1.0), emphasizing the evolutionary stability of the plastome. Comparative analysis of IR boundaries using IRscope revealed a high degree of structural conservation among Pseudolysimachion species, with minor variations at the junction sites. Phylogenetic analysis based on 18 complete plastomes strongly supported the monophyly of the genus Pseudolysimachion (Bootstrap = 100%) and placed P. pyrethrinum var. gasanensis as a sister to the European P. spicatum. These genomic resources provide a foundational tool for the molecular breeding, systematic conservation, and sustainable utilization of this endemic variety, while offering clarity to its taxonomic classification within the tribe Veroniceae.

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