Abstract In this study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to assess cytosine methylation levels in alfalfa (Medicago spp) roots exposed to increasing Naci concentrations (0.0, 8.0, 12.0, and 20.0 dS/m). Eleven indigenous landraces were analyzed, in addition to a salt-tolerant genotype that was used as a control. There was a slight increase in DNA methylation upon exposure to high levels of soil salinity. Phylogenetic analysis using MSAP showed epigenetic variation within and between the alfalfa landraces when exposed to saline conditions. Based on MSAP and enzyme-linked immunosorbent assay results, we found that salinity increased global DNA methylation status, particularly in plants exposed to the highest level of salinity (20 dS/m). Quantitative reverse transcription-polymerase chain reaction indicated that this might be mediated by the overexpression of methyltransferase homolog genes after exposure to saline conditions. Differential genome-wide DNA methylation quantification was carried out by bisulfide conversion and Illumina next-generation sequencing technologies using DNA samples extracted from root tissues exposed to normal conditions and to salinity stress. Around 50 million differentially methylated sites (DMSS) was recognized, 15% of which were significantly (p <0.5) altered in response to salinity. The analysis showed that 77.0% of the DMSs contexts were mCHH while only 9.1% and 13.9% were occupied by the mCHG and mCG contexts, respectively. There was a considerable increase in the DNA methylation levels in all sequence contexts, ranging from 11.7% to 15.3% due to salinity stress. This information was confirmed by mass spectrometry analysis, where the results showed there was a substantial increase of about 19.4% in the total 5mdC levels in response to salinity treatment. Few genes have their DMSs simultaneously distributed along the different structures of a gene; however, there was a contrariwise relationship between the mCG and the mCHH methylation profiles in the promoter and exon regions of the top 2000 DMSs when plants were grown under saline · That is, mCG was mostly hypomethylated but mCHH was hypermethylated in the promoter regions, and mCG contexts were mainly hypermethylated in the exon regions, while mCHH was predominantly hypomethylated in the same region. Ion analysis revealed the presence of a high level Nat and Ç1-ions in the root and leaf tissues of alfalfa exposed to salinity treatment indicating a low capacity for the root system to exclude salts. In addition, the concentration of the proline amino acid has significantly increased in these tissues and the a of proline to the growth media has enhanced, to a certain extent, the salinity tolerance of alfalfa seedlings. Since some of the MSAP markers characterized in this study were coded for sinorhizobium genes, the differential endophytic microbial enrichment upon exposure to salinity was investigated in alfalfa roots. As a prelude to the isolation and utilization of these bacteria in Caliph medic farming, 41 bacterial strains were identified in this project from within the interior of the roots of this plant by pyrosequencing of the small ribosomal subunit (16S rRNA). In addition, the differential abundance of these bacteria was studied following exposure of the plants to salinity stress About 29,064 high-quality reads were obtained from the sequencing of six libraries prepared from control and the salinity-treated tissues. Statistical analysis revealed that the abundance of 70% of the strains was significantly (p < 0.05) altered in roots that were exposed to salinity stress.