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Use of AFLP molecular markers for estimating genetic similarity of alfalfa (Medicago sativa L. Sl.) The aim of study was to determine genetic similarity among sixteen alfalfa populations using the AFLP technique. Plant material was selected considering the high genotypic variability. It included populations of different origin: native forms of alfalfa (secondary ecotypes), inbred lines, single hybrids, synthetic populations, varieties and mutants. Analysis was carried out using 10 combinations of primers. It was noticed, that in case of the studied alfalfa populations, the highest polymorphism was obtained using primers with three selective nucleotides. The genetic similarity coefficients were calculated by the method of Nei and Li. Those coefficients were used for the hierarchical grouping of objects by the unweighted pair group method of arithmetic means. Results of the grouping were presented in the form of dendrogram. Values of coefficients ranged from 0.3311 (between the inbred F line and the Ulstar variety) to 0.8076 (between ecotype 27 and the synthetic population Syn 7-3). Dendrogram of genetic similarity obtained in this way did not fully correspond with the known data about the studied populations' origin. Considering the genetic similarity, inbreed line F was the most outlying from the studied populations of alfalfa.