Due to its very important agronomic value and nutritional quality, Medicago sativa L. is considered as the queen of fodder and the first cultivated forage crop in the world. In field conditions, M. sativa is exposed to several biotic and/or abiotic constraints that affect its quality. In this regard, research is still underway to improve M. sativa resistance to many biotic stresses and, in this context, we analyzed the responses of a core collection of 10 varieties of M. sativa to Phoma medicaginis infection. Results from ANOVA showed that most growth parameters exhibited significant differences between the studied varieties. Nevertheless, only the number of healthy leaves among infection parameters varied significantly between the varieties. The local variety Gabès2355 exhibited the highest biomass. Positive correlations were found between the measured parameters. PCA based on the traits showing significant differences among the studied lines showed that the Gabès variety formed a separate group. Cluster analysis revealed that the studied varieties are classified into three major groups. The first group is formed by Gabès2353, the second group is composed of the Californian and El Hamma varieties, and the third group is constituted of the seven remaining varieties. Gabès2355 was the most tolerant to the Pm8 strain of P. medicaginis while Magna601 variety was the most susceptible. These two varieties will be useful to analyze the physiological and genetic determinants for M. sativa tolerance to P. medicaginis infection.