School-based comprehensive sexuality education (CSE) can help adolescents acquire crucial knowledge and skills to achieve their full potential, particularly in low- and middle-income countries with higher rates of negative sexual and reproductive outcomes. While many low- and middle-income countries have developed CSE curricula, little is known about how these are implemented in the classroom. This multi-country mixed-methods study analysed challenges to the implementation of national CSE curricula in schools in Ghana, Kenya, Peru and Guatemala, based on surveys of secondary school principals, teachers and students aged 15–17 years, and in-depth interviews with central and local government, NGOs and youth organisations. In all four countries, inadequate teacher training remained a major hurdle to effective implementation, manifesting in teacher discomfort and inaccurate messaging. This was compounded by a lack of comprehensive teaching resources. CSE classes were focused mainly on biology at the expense of contraception, gender and rights, despite students’ desire for greater coverage of these topics. Teaching methods lacked interactive activities necessary to develop skills and values. Findings offer useful lessons to improve school-level implementation in these and other countries facing similar challenges. Increasing teacher training and distributing comprehensive CSE materials responsive to adolescents’ needs remain priorities in all countries.