This study critically examines the mathematics knowledge of pre-service post-primary mathematics teachers (N = 85) on commencing their Professional Master of Education (Level 9) initial teacher education (ITE) programme across four institutions in Ireland. Given the nature of a consecutive approach to mathematics teacher education, pre-service teachers enter their ITE programme from a variety of undergraduate degree backgrounds and it is largely accepted that their mathematical content knowledge does not change during their teacher qualification programme. This study utilises a paper-and-pencil test to assess participants’ cognitive and conceptual proficiency with curriculum-aligned mathematical content. Between group comparisons in relation to participants’ undergraduate studies and cognitive and conceptual proficiency are also examined. The data suggest that pre-service teachers demonstrate a strong proficiency with Junior Cycle curriculum-aligned content and poor proficiency with Senior Cycle content, irrespective of the curriculum strand. In addition, significant mean differences exist between participants who had undertaken undergraduate degree studies in physics and mathematics in comparison to other degree programmes. Such investigations are essential given the increased focused on developing highly qualified teachers and policy revisions on ITE programmes. This research alerts us to issues within ITE programmes that may be addressed prior to mathematics teachers’ induction into the profession.