Orchardgrass (Dactylis glomerata) is one of the most famous cool-season perennial forage grasses worldwide, which is economically important for the development of animal husbandry and grassland ecological of our country. The aim of this study was to construct a DNA fingerprint of 21 dominant orchardgrass varieties of China using simple sequence repeat (SSR) makers and start codon targeted polymorphism (SCoT) makers. This study was necessary for orchardgrass varieties identification, management and use. Twenty-four of 180 SSR primer pairs and 24 of 80 SCoT primers were screened based on high polymorphic and clear bands. The 24 SSR primer pairs amplified a total of 186 bands (including 175 polymorphic bands and 6 specific varieties bands) among the 21 orchardgrass cultivars, and the rate of polymorphism was as high as 94.03%. The average value of polymorphic information content (PIC) was 0.845. The Shannon’s information index (I) values ranged from 0.2946 to 0.4633, and the Nei’s gene diversity (H) values ranged from 0.4479 to 0.6549. The numbers of identified varieties ranged from 2 to 21. The 24 SCoT primers amplified a total of 321 bands (including 249 polymorphic bands and 6 specific varieties bands) among the 21 cultivars, and the rate of polymorphism was 76.33%. The average value of PIC was 0.907. The values of I ranged from 0.2588 to 0.6329, and the values of H ranged from 0.1695 to 0.4451. The numbers of identified varieties ranged from 1 to 21. And 10 varieties had specific bands by 5 SSR primer pairs and 5 SCoT primers. And a total of selected 37 bands from 5 primers (A01E14, A01K14, B03E14, D02K13 and SCoT23) were used for the DNA fingerprint construction. Each variety had specific DNA fingerprint code in the database, which would contribute to the identification and the intellectual property rights protect of varieties worldwide.