This paper reports on data arising from a project that is seeking to engage with the long-standing challenge that Physical Education Teacher Education has faced – of how to disrupt pre-service teachers’ (PSTs) established attitudes about physical education and (in an Australian context) the broader learning area of Health and Physical Education (HPE). More specifically the project has confronted stereotypical attitudes that have been internationally recognised as a repeated feature of physical education teaching. Challenging such attitudes was an explicit intention of this research and is arguably critical if teacher education is to extend pre-service teachers’ capacities to positively engage with increasingly diverse student populations. This paper reports on data arising from the validation phase in developing the Diversity and Inclusion Scale in Education: HPE (DISEd-HPE) for use in teacher education institutions. This phase involved administration of the DISEd-HPE to two first year cohort groups of pre-service students following a specialist teacher education program in Health and Physical Education at an Australian University, commencing in 2015 (n=108) and in 2016 (n=87). The paper provides essential background to the scale development, details the methodology associated with the validation phase, and reports on data relating to specific stereotypical attitudes that connected with issues of gender equity, cultural diversity, bodies, ability, and sexuality. The research generated evidence of diverse attitudes towards stereotypes amongst this group of pre-service teachers and evidence of gender-based differences in attitudes. Discussion addresses the implications of these findings for teacher education and the value of further research to pursue the use of the DISEd-HPE as a tool for promoting enhanced awareness and understanding of difference and inclusion amongst pre-service teachers.