Introduction: Cytomegalovirus (CMV) infection in pregnancy is the commonest cause of congenital infection worldwide. Primary CMV infection in pregnancy carries a higher risk of fetal transmission compared to non-primary infection. This study aims to determine the cytokines expression in pregnant women with primary and non-primary CMV infections in both types of infection. Methods: This prospective cohort study was conducted at Microbiology Laboratory, Universiti Sains Malaysia (USM) from January 2019 until June 2020. Seventy-four pregnant women with abnormal pregnancy outcomes with positive CMV IgG with or without IgM by electrochemiluminescence assay (ECLIA) were subjected to IgG avidity assay by ECLIA method to discriminate primary and non-primary CMV infection. Later, the sera were subjected to magnetic Luminex multiplex enzyme-linked immunosorbent assay for cytokine analysis to determine their concentrations in both primary and non-primary CMV infection. Cytokines and chemokines tested were IL-12, IL-2, IFN- γ, TNF-α, IL-1β, IL-6, IL-10, IFN- γ, TNF-α, MCP-1 (CCL-2), and IP-10 (CXCL-10). Results: Concentrations of IL-1β, IL-6, and MCP-1 (CCL-2) were significantly elevated in pregnant women with primary CMV infection with the p-values of (0.001, 0.035, and 0.002) respectively. The intensity of IFN-γ, IL-12, and IL-2 were higher in primary CMV infection with the p-values of (0.018, 0.004, and 0.007). Conclusion: The pro-inflammatory cytokines were expressed significantly in pregnant women with primary CMV infection together with MCP-1 (CCL2), showing predominant Th1 response. The low level of cytokines in non-primary CMV infection might be due to the latent state of CMV in a host.