Seabuckthorn (Hippophae rhamnoides L.) is a hardy fruit-producing plant. With medicinal and nutraceutical properties, its fruits, seeds and leaves are enriched with astonishing array of nutrients. Especially, its bark contains vitamins, essential oils and minerals, for which most of the staple crops are deficient. This study was envisaged to isolate the folate pathway HPPKDHPS ortholog from seabuckthorn. The target gene Hydroxymethyldihydropterin pyrophosphokinase–dihydropteroate synthase linked in the pathway of folate biosynthesis was successfully isolated and cloned. The sequences analysis revealed that this novel genomic locus is 2354 bp in size. The coding region is interrupted by a single large intron. The coding sequence is 1539 bp long which is similar to its ortholog in tomato. The newly isolated gene has prominent nucleotide mutations randomly distributed over the entire length of the sequence. Mutations are only of substitution type, and no deletions or insertions are detectable. All the mutations were of non-synonymous type. Expression profile of HrHPPK-DHPS with semiquantitative RT-PCR revealed the higher accumulation of transcripts in leaf and fruits tissues. Phylogenetic reconstruction revealed gene duplication in soybean and Arabidopsis orthologs. A considerable conservation in 3-dimensional structure of folate protein HPPK-DHPS was observed; however, notable differences in substrate binding pockets were also visible. Hence, HrHPPK-DHPS is a novel ortholog isolated from medicinal plant seabuckthorn. © 2016 Friends Science Publishers