Objective: Evaluate the clinical usefulness of cell-free DNA screening (cfDNA screening) in pregnancies with nuchal translucency (NT) between 95th and 99th percentile. Design: Subgroup analysis of a multicenter prospective cohort study Setting: 12 different secondary and tertiary health care institutions in Korea Sample: 7,547 singleton pregnant women with NT between 95th and 99th percentile Methods: All participants were provided with information about aneuploidy screening or diagnostic testing and selected the first tier test after NT assessment. The first tier test included maternal serum screening tests (MSS), cfDNA screening and invasive test (IT). Main outcome measures: First-tier test preference and chromosomal abnormalities in pregnancies with NT between 95th and 99th percentile Results: A total of 7,547 singleton pregnant women were enrolled and 6,717 cases with known pregnancy outcomes were analyzed. Among these, 89 (1.3%) cases showed NT between 95th and 99th percentile. As the first-tier test, 47 (52.8%) cases chose cfDNA screening, 33 (37.1%) cases selected IT, and nine (10.1%) cases underwent MSS. Chromosomal abnormalities were found in five cases (5.6%), including four cases with trisomy 21 (T21) and one with a balanced translocation. No significant chromosomal abnormalities undetected by cfDNA screening were noted in pregnancies with NT between 95th and 99th percentile. Conclusion: cfDNA screening in pregnancies with NT between 95th and 99th percentile may be considered as an acceptable alternative to invasive test for women intending to avoid the risk of miscarriage.