Although efforts are made to integrate evidence-based medicine (EBM) into clinical practice, physicians experience significant barriers to its implementation. The aim of this study is to quantify the barriers that general practice (GP) trainees experience when using EBM in practice. In September 2008, a questionnaire was administered to 140 GP trainees from three Dutch GP Speciality Training Institutes. The questionnaire focused on barriers that GP trainees meet when using EBM in practice. Factor analysis identified components in which barriers exist, and the validity and reliability of the questionnaire were established. After removing four items that did not fit the questionnaire structure, factor analysis identified three relevant components. All three components had similar mean scores, indicating a similar negative influence of these components on the practice of EBM: knowledge/skills (α = 0.72, mean score 2.9 ± 0.8), attitude (α = 0.70, mean score 2.9 ± 0.6), and external factors (α = 0.66, mean score 3.0 ± 0.5). The barrier that trainees experienced most was lack of time to practise EBM. Barriers to the use of EBM were present in three components: knowledge/skills, attitude, and external factors.