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.