This study focused on the development and validation of a three-tier multiple-choice diagnostic instrument about the ecological footprint. Each question in the three-tier test comprised by; (a) the content tier, assessing content knowledge; (b) the reason tier, assessing explanatory knowledge; and (c) the confidence tier that differentiates lack of knowledge from misconception through the use of a certainty response index. Based on the literature, the propositional knowledge statements and the identified misconceptions of 97 student-teachers, a first version of the test was developed and subsequently administered to another group of 219 student-teachers from Primary and Early Childhood Education Departments. Due to the complexity of the ecological footprint concept, and that it is a newly introduced concept, unknown to the public, both groups have been previously exposed to relevant instruction. Experts in the field established face and content validity. The reliability, in terms of Cronbach's alpha, was found adequate (a = 0.839), and the test-retest reliability, as indicated by Pearson r, was also satisfactory (0.554). The mean performance of the students was 56.24% in total score, 59.75% in content tiers and 48.05% in reason tiers. A variety of concepts about the ecological footprint were also observed. The test can help educators to understand the alternative views that students hold about the ecological footprint concept and assist them in developing the concept through appropriately designed teaching methods and materials.