We aimed to assess opinion and preferences of Croatian pregnant women regarding the prenatal screening tests for trisomies. The study was conducted in Zagreb, Split, Cakovec, Nova Gradiska, Gospić and Zlatar. It was organized in the public primary healthcare centers, among the pregnant volunteers during their first visit to an antenatal clinic (7-12 weeks pregnant). The respondents filled anonymous questionnaire reflecting their knowledge and attitudes to the screening options. In total, 437 completed questionnaires were selected. The average maternal age and the level of education differed significantly between the respondents in the respective cities (P < 0.001). Of the respondents with positive attitude towards screening, the majority would prefer the first-trimester combined test (160/219; 73.1%), while 37/219 (16.9%) opted for the second-trimester biochemical screening. The remaining 22/219 (10.0%) would accept only the ultrasound screening. Among the 224 respondents, who would accept the combined first-trimester test, 95 (42.4%) held a college and university degree, whereas among 59 women, who would choose the second-trimester biochemical screening, 14 were highly educated (23.7%). The difference was statistically significant (P = 0.016). The univariate regression analysis showed that age, level of education and previous information were significant variables predictive for the choice of the test; the level of education and previous knowledge remained significant in the multivariate model. The survey has revealed some of the points that should be improved in the future concept of screening program in Croatia. Health professionals should persist to mend women's knowledge about prenatal screening, taking into consideration women's preferences as well.

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