Objective To analyze variables affecting the differences between on‐to‐on and on‐to‐out methods of nuchal translucency measurement. Design Prospective comparison of two methods of caliper placement. Subjects A total of 245 women undergoing transabdominal and 37 women undergoing transvaginal nuchal translucency scanning. Methods Nuchal translucency was measured using both on‐to‐on and on‐to‐out methods of caliper placement on the same frozen images. Differences between the methods were evaluated according to fetal size, nuchal thickness and mode of examination. Regression analysis was performed for the calculation of the expected difference between the methods at various combinations. Results among 1254 singleton pregnancies were used to set 50th, 95th and 97.5th centiles for the on‐to‐on method. The calculated differences were used to set 50th, 95th and 97.5th centiles for the on‐to‐out method. Results The mean difference between on‐to‐out and on‐to‐on nuchal translucency, using transabdominal scanning, was 0.95 ± 0.14 mm. The differences were shown to correlate directly with fetal size and with nuchal thickness. In addition, scans performed transvaginally exhibited a smaller difference between the methods (0.90 mm vs. 0.94 mm; P < 0.05). Conclusion The difference between on‐to‐on and on‐to‐out nuchal translucency measurements may have a considerable effect on the calculated risk for aneuploidy.