Worldwide appreciated beverage,“Arabica”and“Robusta”coffee produced fromCoffea arabicaandC. canephorarespectively, represents a high economical value product. Arabica coffee is generally sold ata higher price than Robusta coffee. As both intentional and accidental adulterations of Arabica withRobusta are frequent, a reliable, inexpensive, and high-throughput adulteration detection technology isrequired for the coffee industry. We developed a DNA-based authentication method that overcomeslimitations of metabolic profiling of Robusta and Arabica coffees. The identification of Arabica andRobusta coffee species, as well as the quantification of their relative proportion in blends were performedby High Resolution Melting (HRM) analysis on green and roasted coffee products. For a more sensitivedetection method, chloroplastic rather than nuclear genetic variations were targeted betweenCoffeaspecies. By the developed method, theCoffeaspecies of origin can be quickly differentiated and in case ofadulteration, the percentage can be determined