For restructuring educational processes and institutions toward Sustainable Development, teachers’ knowledge and competences are crucial. Due to the high relevance of teachers’ content knowledge, this study aimed to (i) assess Sustainable Development-relevant knowledge by differentiating between situational, conceptual and procedural knowledge, (ii) find out via item response theory modelling how these theoretically distinguished knowledge types can be empirically supported, and (iii) link the knowledge dimension(s) to related constructs. We developed a paper-and-pencil test to assess these three knowledge types (N = 314). A two-dimensional structure that combines situational and conceptual knowledge and that distinguishes situational/conceptual knowledge from procedural knowledge, fits the data best (EAP/PV situational/conceptual: 0.63; EAP/PV procedural: 0.67). Student teachers at master level outperformed bachelor level students in situational/conceptual knowledge but master level students did not differ from students at bachelor level regarding procedural knowledge. We observed only slight correlations between the two knowledge dimensions and the content-related motivational orientations of professional action competence. Student teachers’ deficits in procedural knowledge can be attributed to the small number of Education for Sustainable Development-relevant courses attended. Systematically fostering procedural knowledge in teacher education could promote achieving cognitive learning objectives associated with Sustainable Development Goals in the long term.