The importance of well elaborated cognitive structures in a science knowledge domain has been noted in many studies. Therefore, the main aim of this particular study was to employ a new diagrammatic assessment approach, students' generated systemic synthesis questions (SSynQs), to evaluate and compare the quality of high school students' and pre-service chemistry teachers' cognitive structures in the organic chemistry domain. We used a mixed research sample (N = 83), and SSynQs were constructed following the high school chemical curriculum in the Republic of Serbia. Besides the overall quality of the cognitive structures, the size (extent) and strength (complexity) of the conceptual structures, as external representations of cognitive structures, were also analysed. It was found that both high school students and pre-service chemistry teachers had a substantial size of the conceptual structures, showing relatively good knowledge about IUPAC naming and structures of organic compounds, except for ethers. However, the strength of the conceptual structures, or inter-correlations between organic chemistry concepts, was evaluated as weak within high school students, and medium within pre-service chemistry teachers. This resulted in the identification of three main learning difficulties (LDs), accompanied by a lack of understanding (LU) about the chemical properties and relations of organic compounds. It was surprising to find that all identified LDs and LUs within high school students also appeared within pre-service chemistry teachers. What is more, the most desired and expected cognitive structures (distinguished multidimensional cognitive structures) without LDs and LUs appeared within high school students.