To improve early detection of autism spectrum disorder in preventive care, a Dutch guideline was developed 5 years ago. The guideline provides preventive care physicians at well-baby clinics action-oriented advice and describes a step-by-step approach for children identified at an increased risk for autism spectrum disorder during general healthcare surveillance. This qualitative study evaluated the guideline adherence and studied barriers regarding early detection of autism spectrum disorder at well-baby clinics. Semi-structured interviews were undertaken with 12 preventive care physicians (one representative per province). Interviews were analyzed using grounded theory. It was found that the vast majority of participants did not follow-up general surveillance with an autism spectrum disorder-specific screener as prescribed by the guideline. Six barriers, to be divided in personal, guideline-related and external factors, were found regarding early detection of autism spectrum disorder and adherence to the guideline. The results of this study highlight the importance of an integrative approach, raising awareness of the benefits regarding early detection of autism spectrum disorder in preventive care, the need of continuous investment in easy and accessible training and active screening, and a closer collaboration between preventive care organizations and autism spectrum disorder experts.