A job analysis was conducted to define the knowledge that may be important for entry-level biology teachers. The results will be used to guide the development of new test content specifications for the Praxis Series Biology: Content Knowledge (BCK) assessment. A draft domain was constructed from existing test specifications, national standards, state standards, major biology textbooks, and feedback from Praxis biology standing committee members. A national advisory committee (NAC) of biology teachers and college faculty reviewed and modified the draft domain to reflect the knowledge they believed was important for safe and effective practice and needed at time of entry into the profession. The revised domain consisted of 73 statements clustered within six major categories: (a) Nature of Science: Scientific Inquiry, Methodology, Techniques and History, (b) Molecular and Cellular Biology, (c) Genetics and Evolution, (d) Diversity of Life and Organismal Biology, (e) Ecology: Organisms and Environments, and (f) Science, Technology, and Social Perspectives. Survey responses of 502 biology educators provided independent evidence of the importance of the domain. Respondents judged each knowledge statement on a scale ranging from 1 (not at all important) to 5 (extremely important). Sixty-three knowledge statements had mean ratings above 3.50 in the aggregate sample and for subgroups containing more than 30 respondents. These results indicate that these 63 statements may be considered appropriate for guiding the development of new test content specifications for the Praxis BCK assessment.