In the analysis of the South African NSC diagnostic reports, the Department of Education prepared areas of learners’ struggle have proved to repeat themselves from 2017 to 2020. The classroom practices of Life Sciences teachers, focusing on teacher knowledge, were observed in a sample of schools in the province of Gauteng, South Africa. The investigation aimed to investigate these practices of Life Sciences teachers and consequently diagnose teaching difficulties. Data was collected through interviews and observations in a multi-case study approach and analyzed using Mudau’s (2016) Classroom Practice Diagnostic Framework. This study reveals that teachers’ classroom practices contribute to the teaching difficulties they experience when considering teachers’ knowledge of the Life Sciences content, the teaching context, and learner understanding.