Sainfoin is a very important legume forage that is often planted in arid or semi-arid regions. As herbivorous animal husbandry develops, so does the requirement for sainfoin. However, drought is the main factor limiting the growth of sainfoin. The objective of this study is to explore the response mechanisms of sainfoin varieties/lines to drought stress, which provides the theoretical basis for tolerant breeding of sainfoin. In this study, twenty sainfoin varieties/lines were used as the test materials, the effects of drought stress on growth traits, chlorophyll concentration, leaf water content, osmotic adjustment substances, and antioxidant enzyme activities were determined under simulated levels of drought (− 0.5, − 1.0, − 1.5, and − 2.0 MPa) × exposure times (7 days) in pots. The results showed that leaf area, leaf relative water content, root-shoot ratio, root length, root surface area, root volume, root diameter, root tip number, chlorophyll a content, chlorophyll b content, and catalase activity decreased with increasing drought stress, whereas proline, soluble sugar, soluble protein, and malondialdehyde contents increased in response to drought. Root activity, superoxide dismutase, and peroxidase activities increased first and then decreased and reached the maximum at − 1. 0 MPa. Cluster analysis showed that 20 sainfoin varieties/lines could be divided into 3 categories: (1) High tolerance: new line of P1, Onobrychis viciaefolia ‘Gansu’, code of 1626, and the new line of P2; (2) Moderate tolerance: code of 2323-1, the new line of P3, codes of 2323-2, 2208, 2559, 1668, 7456, 13523, 9977, 13709, 03-132 and 1; and (3) Low tolerance: codes of 2049, 9602, 6, and 7. The findings of these results will help to select the most suitable materials for sainfoin and provide some theoretical basis for cultivating new varieties.