<p>Buckwheat (<i>Fagopyrum</i> species) is an underutilized pseudocereal known for its nutritional and pharmaceutical properties, particularly its high flavonoid content. However, limited information on the genetic and biochemical pathways underlying flavonoid biosynthesis restricts its use in targeted breeding and nutritional enhancement. This study aimed to identify buckwheat species using nuclear and chloroplast DNA barcoding and to investigate the biochemical and genetic basis of flavonoid biosynthesis. Nine morphologically distinct genotypes were analyzed using three molecular markers: ITS (Internal Transcribed Spacer), accD (acetyl-CoA carboxylase beta subunit), and psbA-trnH (photosystem II protein D1 intergenic spacer). Biochemical profiling was conducted along with spatiotemporal gene expression analysis of two key genes involved in the flavonoid pathway: Phenylalanine ammonia lyase (PAL) and Flavonol synthase (FLS). DNA barcoding successfully differentiated the nine genotypes, and biochemical analysis confirmed that the seeds of <i>Fagopyrum tataricum</i> (FT) contain a higher concentration of flavonoids compared to <i>Fagopyrum esculentum</i> (FE). Gene expression analysis across five tissues (root, pre-flowering leaf, post-flowering leaf, flower, and seed) showed significantly higher expression levels of PAL and FLS in FT, supporting the biochemical data. This integrated approach combining DNA barcoding with gene expression profiling enabled accurate species identification and revealed that FT possesses higher flavonoid content. These findings contribute valuable insights into the molecular basis of flavonoid accumulation and lay the groundwork for future genetic engineering or molecular breeding strategies aimed at enhancing the nutritional and pharmaceutical value of buckwheat, promoting its utilization in functional foods and health-related applications.</p>