Orchidaceae, one of the most diverse families of flowering plants, represents an evolutionary masterpiece of adaptation and beauty. Despite their ecological significance and economic value, orchids pose formidable challenges for species-level identification owing to their morphological similarity, hybridization, and phenotypic plasticity. DNA barcoding—a molecular approach using short, standardized DNA regions—has revolutionized plant taxonomy by providing a universal system for rapid and reliable species identification. This chapter reviews the development, principles, and methodologies of DNA barcoding in orchids, emphasizing its role in species discrimination, conservation biology, authentication of medicinal species, and trade regulation. Key barcode markers such as rbcL, matK, ITS2, and trnH–psbA are evaluated for their performance and complementarity. Case studies from Asia and Europe illustrate its application in authenticating herbal materials, monitoring illegal trade, and resolving cryptic taxa. The chapter also discusses challenges such as low interspecific divergence, hybridization, and incomplete reference libraries, while highlighting advances in multilocus and next-generation sequencing (NGS) approaches. DNA barcoding stands as a bridge between traditional morphology and modern genomics—an indispensable tool for the taxonomy, conservation, and sustainable use of Orchidaceae.