A cytological investigation was conducted on the 10 Chinese tetraploid alfalfa （Medicago Sativa L.）germplasm accessions from the different regions using a technique of fluorescence in situ hybridization (FISH). Three different repetitive sequences were adopted as FISH probes , which are 5S rDNA、45S rDNA and C0t-1 DNA. The result showed that 32 chromosomes of alfalfa could be characterized as 16 pairs with the above probes combination, and the FISH patterns among the different materials revealed high similarity. Chromosome polymorphisms were uncovered both within and among genotypes. Chromosome 1 (satellite chromosome) with 7 variant showed to be highest polymorphic, while 3 homologue chromosomes showed to be more conserved and monomorphic. Chromosome polymorphisms were compared among 10 alfalfa germplasm accessions geographically distributing from western to eastern regions of China. The result showed that a few chromosome variants in germplasm accessions from Tibet, Xinjiang, and Liaoning are distinct from those in the others.