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Alfalfa (Medicago sativa L.) is a forage legume of world-wide importance used in agriculture. The genetic relationship and distance among cultivars is of great interest for breeding programs. Random amplified polymorphic DNA (RAPD) was used in the current study to evaluate genetic variability of 7 alfalfa cultivars in Northwest China. A total of 132 discernible loci were obtained for all populations using 10 primers, and 88.64% of these loci were polymorphic, which indicated that a high diversity existed in the cultivars from Northwest China. Analysis of molecular variation (AMOVA) showed that the majority of the genetic variation was within cultivars (60.4%), with a relatively smaller proportion being due to the differences between cultivars (39.6%). The smallest genetic distance (0.0813) was estimated between cultivars Gannong-2 and Zhonglan-1, while the largest (0.2840) was between cultivars Gannong-1 and Tianshui. Cluster analysis using the UPGMA method based on Nei's similarity coefficient divided studied populations into five groups. The RAPD-derived diversity data were in correspondence with habitat heterogeneity of 7 alfalfa cultivars in Northwest China, which suggesting that alfalfa cultivars in Northwest China tended to be divergent to adapt to different stress environments. [ABSTRACT FROM AUTHOR]