The objective of the present study is to analyze pre-service teachers’ mathematics knowledge base as a relationship between their substantive subject mathematics knowledge and syntactic subject mathematics knowledge. The theoretical approach refers to Shulman’s pedagogical content knowledge and Ball, Thames and Phelps’ subject matter knowledge for the teaching concept. The participating pre-service teachers (PSTs) were divided into two groups: 20 from a program course for K–3 teachers and 25 from a program course for grade 4–6 teachers. The crucial mathematical content for the study was algebraic patterns, rational numbers (fractions), proportionality and elements of combinatorics. The findings show the importance of gaining a better insight into and understanding of student teachers’ learning process and challenges related to the mathematics knowledge base (e.g., its substantive and syntactic components). The benefits of the empirical findings are that they led to new knowledge and a deeper understanding of PSTs’ learning of mathematical concepts through two learning processes (SUB and SYN), as well as the relationship and shift between these processes.