Abstract Objective to assess the knowledge of cytomegalovirus (CMV) infection among Dutch primary care midwives, and clinical approaches to informing women about CMV. Design cross-sectional study, using self-administered questionnaires. Participants 330 Dutch primary care midwives. Setting primary midwifery care practices across the Netherlands. Main outcome Midwives׳ knowledge of CMV transmission routes and maternal symptoms, and clinical practice behaviours regarding CMV, the information typically provided or reasons for not informing pregnant women about CMV. Findings the overall median knowledge score was 8.0 out of a maximum possible score of 13.0. Of all participants, 10.6% reported always informing pregnant women about CMV infection prevention and 41.0% reported never informing pregnant women. The main reason indicated for not informing pregnant women was lack of knowledge about preventive methods (45.7%). Conclusion Dutch primary care midwives have limited knowledge of CMV infection. Improvement in providing education to pregnant women about strategies to prevent CMV is necessary.