With the development of intelligent automotive products, in-vehicle ambient lighting has received increasing attention, as shown by the growing number of models on the market with in-vehicle ambient lighting installed and the growing range of ambient lighting installation positions. Currently, there is no industry guideline for the appropriate position of ambient lights. Therefore, this study investigated user preference for 15 ambient light positions and explored whether driving experience, demographic variables, and ambient light usage experience influence user preference for ambient light positions. This study used two indicators to measure users' preference for ambient light positions, aesthetic evaluation and favourite ranking. The results of 479 valid questionnaires showed that users’ preference for 15 ambient light positions can be divided into three categories. The ambient light positions with high preference were those surrounding the centre screen, armrest and centre screen bottom. Users had a low preference for the upper front windshield, steering wheel, bottom of left and right windows, A-pillar and rear view mirror. It was also found that users' aesthetic evaluation of each position was influenced by gender and experience with ambient lighting. Male drivers, users with ambient lights currently installed in their vehicles and users with more frequent use of ambient lights showed a higher aesthetic evaluation for each position, indicating a more open attitude toward ambient lighting. The results of this study can provide a reference for the layout of in-vehicle ambient lighting, help relevant practitioners determine in-vehicle ambient light positions that better meet user needs, and suggest that ambient light layouts should be designed to better match the characteristics of consumer groups.