Teaching mathematics requires a wide range of knowledge, including types of mathematical knowledge specialized to the work of teaching. Because specialized content knowledge is a form of professional knowledge, it is important to emphasize in professional preparation. In this paper, we present results comparing the performance of prospective and practicing teachers on the Learning Mathematics for Teaching (LMT) assessments. While the results show that practicing teachers score higher on the LMT assessments than prospective teachers, only about half of the individual items are significantly more difficult for prospective teachers. To gain a more nuanced understanding of the mathematical knowledge assessed by the LMT items, we also compared the performance of prospective and practicing teachers for items organized by the mathematical work of teaching. We discuss implications for identifying types of professional mathematical knowledge important to emphasize in teacher education.