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The present study was designed to evaluate the gene expression and DNA methylation level in the promoter region of the <i>CD4</i> and the JAK-STAT-pathway-related genes. A total of 24 samples were deployed in the gene expression and 118 samples were used in the DNA methylation study. Student’s <i>t</i>-tests were used to analyze the gene expression and DNA methylation. The evaluation of DNA methylation in promoter regions of <i>JAK2</i> and <i>STAT</i><i>5A</i> revealed hypo-methylation levels of CpG sites and higher gene expression in cows diagnosed with mastitis as compared to the healthy control, and vice versa in those with <i>CD4</i>. DNA methylation was negatively correlated with gene expression in <i>JAK2</i>, <i>STAT</i><i>5A</i>, and <i>CD4</i> genes. Six, two, and four active transcription factors were identified on the CpG sites in the promoter regions of <i>JAK2</i>, <i>STAT</i><i>5A</i>, and <i>CD4</i> genes, respectively. Regarding correlation analysis, the DNA methylation levels of <i>CD4</i> showed significantly higher positive correlations with somatic cell counts (<i>p</i> < 0.05). Findings of the current study inferred that aberrant DNA methylation in the CpG sites at the 1 kb promoter region in <i>JAK2</i>, <i>STAT</i><i>5A</i>, and <i>CD4</i> genes due to mastitis in cows can be used as potential epigenetic markers to estimate bovine mastitis susceptibility in dairy cattle.