Juncus (Juncaceae) comprises over 300 species with high morphological plasticity, and its systematics remain incompletely resolved due to limited genomic resources. Here, we generated complete plastid genomes for two Korean
Juncus species (
J.
decipiens and
J.
gracillimus) and incorporated plastid coding genes from an additional species to reconstruct phylogenetic relationships and examine plastome evolution within Juncaceae. Comparative analyses revealed substantial plastome size variation across
Juncus and
Luzula, largely driven by changes in inverted repeat (IR) length, with
Luzula plastomes showing pronounced IR expansion. Within
Juncus, extensive structural rearrangements were detected, including multiple inversion events, and closely related taxa shared conserved inversion patterns. Phylogenomic analyses recovered well-supported clades that were associated with structural traits such as extreme small single-copy (SSC) contraction and consistent loss of the plastid
ndh, some
rps and
rpl gene families, indicating clade-specific plastome evolution in Juncaceae. To support applied molecular identification, we identified
J.
decipiens-specific plastid diagnostic SNPs (
matK,
rpl2) and validated allele-specific PCR markers using individuals from different species within the
Juncus genus. In parallel, transcriptome sequencing of
J.
decipiens generated 133,559 transcripts and 66,324 unigenes, enabling discovery of high-confidence nuclear exonic SNP loci by mapping reads to a
J. effusus nuclear genome. Collectively, our results provide new insights into plastome structural evolution and gene loss in
Juncus and deliver validated plastid and nuclear markers for authentication and future conservation or utilisation studies on
J.
decipiens.
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