Breast cancer is one of the most frequent cancers among women today. Interleukine-35 (IL-35), interleukine-38 (IL-38) and matrix metalloproteinase-9 (MMP-9) have been reported to play a potential role of tumor development and aggressiveness. However, few studies have compared the expression of IL-35, IL-38 and mRNA MMP-9 between primary breast cancer and benign breast tumor. Therefore, this study was aimed to investigate and compare the preoperative level of serum IL-35, IL-38 and the expression of mRNA MMP-9 of human breast cancer with benign breast tumor. In addition, elucidate the possible role of IL-35, IL38 and MMP-9 as prognostic markers for breast cancer and correlate it’s with the clinicopathological factors. A total of 90 Iraqi women including 60 patients with breast tumor (30 with primary breast cancer and 30 with benign breast tumor) and 30 healthy women were enrolled in the study. Blood and tissue sampling was done after taking a written consent. Preoperative serum level of IL-35 and IL-38 were measured for all participants by using human enzyme-linked immunosorbent assay (ELISA). The level of mRNA MMP 9 expression was determined in 30 patients (20 with primary breast cancer and 10 with benign breast tumor) by using real time reverse transcription-polymerase chain reaction (RT PCR). In addition, the clinicopathological factors of all the patients were collected. The result showed that serum IL-35 levels of patients with primary breast cancer was significantly higher compared with that of patients with benign breast tumor and in the healthy control group (P<0.001). However, there was no significant difference in the serum IL-38 levels between patients with primary breast cancer, benign breast tumor and the healthy control group (P=0.78). The area under curve (AUC) of IL-35 and IL-38 for distinguishing patients with primary breast cancer and healthy control subjects was 1.000 (95% CI: 0.940 to 1.000) and 0.756 (95% CI: 0.628 to 0.858) respectively. High level of IL-35 was significantly correlated with lymph node metastasis, and in patients with age ≥45 and with no family history for breast cancer (P<0.05), whereas IL-38, revealed a significant positive correlation with patients age ≥45, postmenopausal women and lymph node metastasis. The mRNA MMP-9 expression level was significantly raised in patients with primary breast cancer compared to the patients with benign breast tumor. A significant rise in mRNA MMP-9 expression was found in patients presenting with lymph node metastasis. In conclusion, preoperative levels of IL35 exhibit higher accuracy for primary breast cancer than IL-38. Our results suggest that serum IL-35 level and mRNA MMP-9 expression level are better markers than serum IL-38 in predicting breast cancer development and progression.