Bacterial wilt caused by Clavibacter michiganensis subsp. insidiosus (Cmi) is an integral part of chronic vascular wilt of alfalfa. The aim of the presented study was to determine the virulence of Cmi strains and later on to evaluate the level of resistance of alfalfa varieties registered in the Czech Republic and of some standard check varieties. Three methods of inoculation were compared at greenhouse conditions. The severity of the disease was determined on the basis of symptoms appearing on the cross section of the roots. The Cmi strain 12/5/98 and its re-isolates showed a high level of virulence. Freeze-dried isolates lost their virulence. The best differentiation of tested varieties according to the level of resistance was obtained by using successive inoculation on cotyledons, stems and roots. Of tested alfalfa varieties, Vernal and Roamer were classified as resistant and Nitranka, Orca and Europe as the most susceptible. Alfalfa varieties (Czech and European) registered in the Czech Republic recently proved lower disease severity in comparison with ones registered formerly (r = –0.538, p (0.05)=0.553). The above mentioned fact evidences the progress of Cmi resistant alfalfa breeding in Europe.