Misalignments in teaching pedagogies between secondary schools and tertiary institutions have exacerbated educational disparities among students from different backgrounds. Given the variation in students' educational background and competencies, there was a need to develop an Anatomy and Physiology (A&P) Foundational Knowledge Assessment to establish the levels of preparedness of first-year medical students. Previous work that focused on the development of the assessment showed it to be effective in measuring students' foundational knowledge in human anatomy and physiology. The aim of this study was to assess the validity of the A&P Foundational Knowledge Assessment in determining students' prior knowledge and predicting academic performance of first-year students in their anatomy and physiology studies. Three hundred seventy first-year students, across two cohort years, 2017 and 2018, completed the A&P Foundational Knowledge Assessment. Data were analyzed through descriptive statistics, analysis of variance, and Pearson's correlation. Results show that for both cohorts [approximately]30% of students scored [less than or equal to]55% and were potentially at risk of performing poorly in their anatomy and physiology studies. Pearson's correlation showed a significant relationship between students' performance on the foundational knowledge assessment and their anatomy and physiology assessments. For both cohorts, >10% of students identified by the A&P Foundational Knowledge Assessment were at risk of either failing the course, entering an extended degree program, or being excluded from the program. Results indicate that the assessment is a good predictor for differentiating medical students' performance in first-year anatomy and physiology.