This study investigates challenges in understanding the mole concept among pre-service teachers in Ghanaian Colleges of Education. Chemistry education is fundamental for fields such as medicine and engineering, yet students often face difficulties due to inadequate foundational knowledge and ineffective teaching methods. The mole concept is crucial for grasping various chemical phenomena, but its complexity frequently results in significant learning challenges. This study employs a mixed-method explanatory design, combining quantitative diagnostic tests and qualitative interviews with 1,211 pre-service teachers from six selected colleges. The study identifies key challenges in learning the mole concept, including difficulties in translating word problems into equations, determining mole ratios, deriving empirical and molecular formulae, and understanding the relationship between stoichiometric coefficients and moles. The analysis categorizes these challenges into comprehension, transformation, process skills, and encoding errors, with process skills and transformation challenges being the most prevalent. Findings reveal that many students struggle to solve problems without formulae, highlighting a lack of conceptual understanding. Interviews with pre-service teachers confirm quantitative data, indicating that students often rely on rote memorization rather than conceptual grasp. The study concludes that improvements in teaching methods are needed, emphasizing the importance of understanding over memorization and recommending small group collaborative learning. Contributions include filling empirical gaps in the literature regarding pre-service teachers’ challenges and suggesting practical recommendations for enhancing chemistry education.