Spring black stem and leaf spot, caused by <i>Phoma medicaginis</i>, is an issue in annual Medicago species. Therefore, in this study, we analyzed the response to <i>P. medicaginis</i> infection in a collection of 46 lines of three annual Medicago species (<i>M. truncatula</i>, <i>M. ciliaris</i>, and <i>M. polymorpha</i>) showing different geographic distribution in Tunisia. The reaction in the host to the disease is explained by the effects based on plant species, lines nested within species, treatment, the interaction of species × treatment, and the interaction of lines nested within species × treatment. <i>Medicago ciliaris</i> was the least affected for aerial growth under infection. Furthermore, the largest variation within species was found for <i>M. truncatula</i> under both conditions. Principal component analysis and hierarchical classification showed that <i>M. ciliaris</i> lines formed a separate group under control treatment and <i>P. medicaginis</i> infection and they are the most vigorous in growth. These results indicate that <i>M. ciliaris</i> is the least susceptible in response to <i>P. medicaginis</i> infection among the three Medicago species investigated here, which can be used as a good candidate in crop rotation to reduce disease pressure in the field and as a source of <i>P. medicaginis</i> resistance for the improvement of forage legumes.