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Leymus chinensis (Trin.) Tzvel is a perennial grass in the tribe Gramineae and important forage in Northern China. Knowledge of its genetic diversity is a prerequisite for using modern breeding techniques. Amplified fragment length polymorphism (AFLP) was first used to evaluate the genetic relationship among and within three ecotypes. Distinct clusters were produced based on AFLP markers. All accessions from the same ecotype were grouped in a cluster except accessions 6. According to AFLP profile ecotype-specific bands differ from each other. The genetic differentiation within the ecotype of the species was much smaller than that among ecotypes. Self-incompatibility in this species contributes to evident genetic differentiation together with environment. These results indicate that ecotypes were distinguished visually similarly to genetic variation.