**5. Conclusions**

From the genomic information of *M. sativa*, we discovered a total of 16 *MsHSF* genes in this study. We analyzed the physicochemical properties of the 16 MsHSF proteins and found that all the *MsHSF* genes were localized in the nucleus. Amino acid sequence comparison showed that all *MsHSF* genes contain conserved DBD structures, after which we classified MsHSF proteins into 2 groups and 15 subgroups based on evolutionary relationships and showed that they are mostly similar within the same group but differ significantly between subgroups. Cis-acting element and GO enrichment analyses suggest that *MsHSF* genes may be involved in multiple transcriptional regulatory mechanisms for plant growth and stress response. In addition, expression profiling indicated that *MsHSF* genes could show significant specificity during tissue development and that *MsHSF* genes play a very important role in response to abiotic stresses. Overall, bioinformatic analysis and expression profiling studies of HSF can help to understand the important role of HSF in abiotic stress responses in alfalfa and provide a basis for exploring ways to understand and regulate these stress responses.

**Author Contributions:** Conceptualization, C.G., J.M., G.Z. and Y.Z.; methodology, J.M.; software, G.Z.; validation, Y.Y. and Y.Z.; investigation, J.M. and Y.Y.; data curation, G.Z. and Q.M.; writing original draft preparation, J.M. and G.Z.; writing—review and editing, C.G., J.M., G.Z., S.H. and L.S.; funding acquisition, C.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by Zhejiang Provincial Natural Science Foundation of China (No. LY21C160001); National Natural Science Foundation of China (31272494); and Zhejiang Provincial Natural Science Foundation of China (No. LY16C170003).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data in this study can be found in the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

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