We used an integrated morpho-physiological, biochemical, and genetic approach to investigate the salt responses of four lines (TN1.11, TN6.18, JA17, and A10) of Medicago truncatula. Results showed that TN1.11 exhibited a high tolerance to salinity, compared with the other lines, recording a salinity induced an increase in soluble sugars and soluble proteins, a slight decrease in malondialdehyde (MDA) accumulation, and less reduction in plant biomass. TN6.18 was the most susceptible to salinity as it showed less plant weight, had elevated levels of MDA, and lower levels of soluble sugars and soluble proteins under salt stress. As transcription factors of the APETALA2/ethylene responsive factor (AP2/ERF) family play important roles in plant growth, development, and responses to biotic and abiotic stresses, we performed a functional characterization of MtERF1 gene. Real-time PCR analysis revealed that MtERF1 is mainly expressed in roots and is inducible by NaCl and low temperature. Additionally, under salt stress, a greater increase in the expression of MtERF1 was found in TN1.11 plants than that in TN6.18. Therefore, the MtERF1 pattern of expression may provide a useful marker for discriminating among lines of M. truncatula and can be used as a tool in breeding programs aiming at obtaining Medicago lines with improved salt tolerance.