Nursing students struggle to retain enough anatomical knowledge to meet their entry to practice competencies, but what knowledge is missing and when this occurs has been previously unexplored. A cohort of 80 nursing students were given multiple choice quizzes to assess their anatomical knowledge on 11 different organ systems during their second, third, and fourth year. Results were analyzed in comparison to their first-year examination scores to determine knowledge loss. Results showed an overall knowledge loss of 33.5% in the second year, 31.8% in the third year, and 29.6% in the fourth year. There were significant differences in system specific results. Special senses (i.e., audition) had a 20.6% loss in the second year, increased in retention to a 17.3% loss in third year, and then decreased to a 37% loss in fourth year. The vascular system had a 46.1% knowledge loss at the second-year assessment, declined to 49% knowledge loss in the third year, but improved to 27.6% knowledge loss by the fourth year. A similar change was observed for the musculoskeletal system with second-year loss at 30.7%, third-year loss at 40.3%, and fourth-year loss at 26.6%. These data suggest there are significant differences in the amount of knowledge retained by nursing students depending on the system being tested and the year the test is taken. Identifying the areas and times where anatomical knowledge is lost and gained is valuable for instructors in any program so that specific topics can be targeted at different times with more effective educational strategies.