OBJECTIVES: The aim of this study was to assess the seroprevalence and associated factors of cytomegalovirus (CMV) among pregnant women in Southern Ethiopia. DESIGN: Cross-sectional study. SETTING: The study was conducted in Hawassa University comprehensive and specialised hospital. Hawassa, Southern Ethiopia. PARTICIPANTS: A total of 600 consecutive pregnant women attending the delivery ward were recruited for the study from August to October 2020. OUTCOME MEASURES: The study assessed the rate of maternal anti-CMV IgG and IgM antibodies. The association of obstetric history, sociodemographic and behavioural characteristics with seropositivity of CMV was also evaluated based on the collected data using structured questioners. RESULTS: Seropositivity for CMV IgM antibodies was 8.2% (49/600) (95% CI 6% to 10.5%), whereas the CMV IgG was 88.7% (532/600), (95% CI 89.5% to 94.0%). Seroprevalence of CMV IgM was higher in women of older age, currently unmarried, having nursery schooled children and with any of the detected curable sexually transmitted infections, while seroprevalence of CMV IgG was significantly associated only with women having nursery schooled children. Seroprevalence was not significantly associated with previous adverse pregnancy outcome, gravidity, being a child daycare occupant mother and newborn birth weight. CONCLUSION: In the present study, we identified a high rate of CMV IgM and CMV IgG seroprevalence among pregnant women in Southern Ethiopia. Given that there is no existing CMV diagnosis, special attention should be designed to pregnant women in parallel to the existing antenatal care facility. Besides, training healthcare professionals will support awareness conception among pregnant women concerning the sequels of CMV infection during pregnancy.