Holistic and pluralistic instructional beliefs and instructional practices in Education for Sustainable Development are often considered of high importance to foster students' competences. This mixed-method study describes the development and the validation of the Holism-Pluralism in Education for Sustainable Development questionnaire (HPESD-Q) to measure teachers' instructional beliefs and their instructional practices in ESD. The development process includes a survey to choose participants for interviews, review from an expert team, cognitive testing, pilot-testing and the main-run study. Confirmatory factor analysis, reliability and intercorrelations analysis were applied in the validation process. The final version of the HPESD-Q consists of the Instructional Beliefs scales and the Instructional Practices scales. Both have good fit-of-goodness indicators (CLI = 0.992, TLI = 0.9891, RMSEA = 0.030 and CLI = 0.984, TLI = 0.980, RMSEA = 0.043, respectively). The Cronbach's [alpha] for the scales range from 0.90 to 0.97, indicating good internal consistency, while the intercorrelations indicate good discriminant validity. The results indicate the potential of the questionnaire for research and practice in ESD.