Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are multisystemic autoimmune diseases with multifactorial nature. Considering limitations of the current conventional serological tests for diagnosis of these diseases, researchers strive to find new and more valid biomarkers. The methylation level of interferon-induced protein 44-like (IFI44L) promoter was evaluated in 69 patients with SLE, 61 patients with RA, and 71 healthy subjects. Quantitative methylation of the promoter region of IFI44L gene was measured in DNA extracted from peripheral blood mononuclear cells (PBMCs) with methylation-quantification endonuclease-resistant DNA (MethyQESD) method. Our findings revealed a substantial hypomethylation of IFI44L promoter in SLE and RA patients compared with healthy volunteers (mean: 60.36%±64.54%, 47.59%±30.34%, and 89.17%±76.96%, respectively; PSLE= 0.018, PRA<0.001). In comparison between SLE and RA patients with control group, IFI44L promoter methylation had a sensitivity of 84/06% and 93/65% and specificity of 32/39% and 29/58, respectively. The promoter methylation level was not meaningfully different between SLE and RA patients (P > 0.05). Moreover, our analysis revealed that the methylation level of IFI44L promoter was not statistically significantly different between SLE disease activity and renal involvements (P > 0.05). While RA patients with a higher concentration of CRP had a lower DNA methylation level (P = 0.012). The methylation level of IFI44L promoter was lower in PBMCs of Iranian patients with SLE and RA than that control group. Furthermore, DNA methylation level of IFI44L promoter had a negative correlation with RA disease activity. However, there was not a significant association between clinical characteristics of SLE.