Background <p>To evaluate the best maternal point of visual focusing (PVF) for minimizing pain and anxiety during amniocentesis.</p> Methods <p>A prospective randomized study was conducted to compare pain and anxiety levels assessed by a visual analogue scale (VAS) on anticipated and perceived pain after amniocentesis between three groups of pregnant women who underwent amniocentesis with three different PVF. Differences between means of anticipated and perceived pain in each maternal PVF were also evaluated.</p> Results <p>A total of 105 participants were enrolled and randomly assigned to covered maternal eyes, independent PVF and adapted visual reality (VR) groups, respectively. The mean VAS scores of anticipated pain and perceived pain were not significantly different between each different maternal PVF. The mean VAS of perceived pain was significantly lower than that of anticipated pain in all maternal PVF groups. The different mean VAS scores (95% confidence interval: CI) were 1.50 (0.28–2.71), p-value 0.017 in the maternal covered eyes group, 1.17 (0.16–2.18), p-value 0.025 in the independent PVF group and 0.96 (0.06–1.86), p-value 0.037 in the adapted VR group.</p> Conclusion <p>Covered maternal eyes, independent PVF and adapted VR can all be similarly recommended as the best maternal PVF during amniocentesis.</p> Trial registration <p>Srinakharinwirot ethics committee (SWUEC/E/M-059/2565) and Thai Clinical Trials Registry TCTRID20221022003 (19 October2022).</p>