This study concerns the importance of assessing teachers’ content knowledge (CK) and it is one of the very few international studies that directly measures science teachers’ CK. We developed a comprehensive content knowledge test (CKT), relating to the content strand: states of matter. The study compared teachers from the Jewish and the Arab sectors in three stages of professional development: Results showed that all participants’ CK level was low (M = 61.3%). Comparison between the sectors showed that CK scores among the Arab pre-service and science teachers were significantly higher than CK scores among the Jewish pre-service and science teachers. Our study is an example of a direct CK assessment of teachers that provides valuable diagnostic information. Its dismal findings illuminate the need for innovative methods to measure teachers’ CK directly in various disciplines, and contributes strong impetus for a renewed focus on the training and professional development of teachers.