Scrophularia ningpoensis, a perennial medicinal plant from the Scrophulariaceae family, is the original species ofScrophulariaeRadix (SR) in the Chinese Pharmacopoeia. This medicine is usually deliberately substituted or accidentally contaminated with other closely related species includingS. kakudensis,S. buergeriana, andS. yoshimurae. Given the ambiguous identification of germplasm and complex evolutionary relationships within the genus, the complete chloroplast genomes of the four mentionedScrophulariaspecies were sequenced and characterized. Comparative genomic studies revealed a high degree of conservation in genomic structure, gene arrangement, and content within the species, with the entire chloroplast genome spanning 153,016–153,631 bp in full length, encoding 132 genes, including 80 protein-coding genes, 4 rRNA genes, 30 tRNA genes, and 18 duplicated genes. We identified 8 highly variable plastid regions and 39–44 SSRs as potential molecular markers for further species identification in the genus. The consistent and robust phylogenetic relationships ofS. ningpoensisand its common adulterants were firstly established using a total of 28 plastid genomes from the Scrophulariaceae family. In the monophyletic group,S. kakudensiswas determined to be the earliest diverging species, succeeded byS. ningpoensis. Meanwhile,S. yoshimuraeandS. buergerianawere clustered together as sister clades. Our research manifestly illustrates the efficacy of plastid genomes in identifyingS. ningpoensisand its counterfeits and will also contribute to a deeper understanding of the evolutionary processes withinScrophularia.