Aim: Different screening strategies have been proposed to detect high risk women during pregnancy for Down syndrome. In order to achieve effective screening, the patient making the choice and the staff offering the test must have a reasonable understanding of all facts relevant to the test. The aim of this study was to assess the existing knowledge and awareness of antenatal Down syndrome screening in patients and obstetric stakeholders across eight major obstetric centres in Sri Lanka. Methods: This was a prospective study carried out between January and June 2013 in eight tertiary care settings in 7 districts representing Northern, Western, Eastern, Southern and central provinces in Sri Lanka. A validated questionnaire was translated from English into Singhalese and Tamil and independently translated back to English and piloted to confirm the accuracy of the translation. This translated questionnaire was distributed among antenatal patients and obstetric unit staff members. Results: A total of 1116 patients and 535 staff members were recruited. Present overall knowledge of Down syndrome among antenatal patients was poor in all 7 districts. Majority of patients were not aware that available options of screening for Down syndrome (Awareness about nuchal translucency-21.6% (95% CI 14.7-30.6%), biochemical screening-26.3% (95% CI 18.7-35.7%) invasive procedures-23.3 (95% CI, 16.1-32.5%). Majority of staff members were also not aware about available screening strategies (Awareness about nuchal translucency-29.3% (95% CI 21.3-38.9%), biochemical screening-26.9% (95% CI 19.2-36.3%) but their knowledge of diagnostic tests were high (invasive procedures-59.4% (49.6-68.5%). Moreover, there is no difference in knowledge in different part of the island. Conclusions: Adequate education on available screening methods of Down syndrome for the staff is a timely need so that the means would be available to disseminate knowledge to the wider patient and public populations.