The present study aimed to determine the knowledge levels secondary school students have on ecosystem, biodiversity, and environmental problems within the context of a science class and to identify the relationship between their knowledge levels on these subjects according to gender and science course grades. The study applied the survey design, a quantitative research method. The participants for the study included 400 eighth-grade students from a public secondary school in Ankara, Turkey during the 2018-2019 academic year. The Ecosystem, Biodiversity, and Environmental Problems Achievement Test (EBET) was used to collect the study data, and SPPS 17 software was used for data analysis. The students' responses to EBET were evaluated based on Bloom's Original taxonomy. The results revealed that the students had no difficulty in answering knowledge and comprehension questions, partial difficulty in answering application questions, and great difficulty in answering analysis questions. There was a positive, moderately significant relationship between the students' science course grades and their scores on the knowledge and comprehension questions; however, no significant relationship was found between their science course grades and their scores on application and analysis questions. Regression analysis was performed to determine the predictive value of science course grades on the knowledge levels of ecosystem, biodiversity and environmental problems. The students' knowledge and comprehension scores varied significantly by gender in favor of the female students, whereas their application and analysis scores did not vary significantly by gender.