This study compares anatomy diagram production skills as a part of Visual Literacy competence in Biology Education in schoolchildren (n = 254) and pre-service teachers (n = 282). Both groups produced cross-sectional anatomy diagrams from knowledge acquired using frontal views and answered a questionnaire on their difficulties with the exercise. Overall, children performed better than teachers. However, both groups scored very low, revealing a lack of Visual Literacy, but also low content knowledge. Participants recognised their difficulty visualising the requested cross-section, especially with the distribution and size of organs. These results provide evidence of the need to improve anatomical knowledge and Visual Literacy skills in teacher training, since future teachers must teach these topics in schools.