Objectives Our hypothesis was that pediatric residents and medical students who participated in a structured forensic evidence collection course would have improved knowledge of prepubertal evidence collection practices and pubertal genital anatomy. Methods The course curriculum included a forensic evidence collection video created by the sexual assault nurse examiner directors. After watching the video, the participants simulated forensic evidence collection using forensic evidence collection kits and chain of evidence protocols in a hybrid simulation setting under the supervision of a pediatric sexual assault nurse examiner. The participants completed a multiple-choice test and a fill-in-the-blank anatomical diagram test before and after the course. Results Of an eligible 48 participants, 42 completed the course; therefore, our participant response rate was 87.5%. There was significant improvement in knowledge, with an average pretest score of 62% ± 20% and the average posttest score of 86% ± 9% (P < 0.001). Qualitative evaluations were overwhelmingly positive, with consistent scoring of 6/6 in a 6-point agree scale. Learning themes, which emerged from open-ended questions on the evaluations, included knowledge gained on evidence collection processes (n = 26), how to appropriately interact with abused patients (n = 8), hands-on nature of the experience and the benefits of walking through the examination (n = 7), and pubertal genital anatomy knowledge (n = 3). Participants suggested that more instruction on anatomy would be helpful. Conclusions We found that pediatric residents' and medical students' knowledge of pediatric sexual abuse may be improved with a short simulation course focusing on forensic evidence collection.