The public is more disconnected from agriculture than ever. Americans are now two to four generations removed from the farm with a majority of Americans having no direct experience in agriculture. As a result, the public lacks the knowledge and appreciation of the food, fuel, and fiber it demands. The National Agricultural Learning Objectives (NALOs) were recently developed to describe students' agricultural knowledge but have, as yet, not been used to guide research into students' agricultural literacy. The purpose of this project is to further understand students' agricultural literacy through NALO-based assessment of students' knowledge. This study focused on the NALOs in the areas of agriculture and the environment (AgE) and the STEM dimensions of agriculture (STEM) using a sequential exploratory mixed methods design. Thirty-five students participated in semi-structured interviews surrounding the NALOs. Interview data were coded and analyzed while using the evidence-centered design process to create empirically grounded assessments that were administered to a sample (n = 400) of elementary students. Results suggest that students are more knowledgeable about the STEM dimensions of agriculture than the agricultural and environmental topics. Recommendations are provided to guide future research and development around the NALOs.