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To excel in modern science, technology, engineering, and mathematics careers, biology majors need a range of transferable skills, yet competency development is often a relatively underdeveloped facet of the undergraduate curriculum. We have elaborated the Vision and Change core competency framework into a resource called the BioSkills Guide, a set of measurable learning outcomes that can be more readily implemented by faculty. Following an iterative review process including more than 200 educators, we gathered evidence of the BioSkills Guide’s content validity using a national survey of more than 400 educators. Rates of respondent support were high (74.3–99.6%) across the 77 outcomes in the final draft. Our national sample during the development and validation phases included college biology educators representing more than 250 institutions, including 73 community colleges, and a range of course levels and biology subdisciplines. Comparison of the BioSkills Guide with other science competency frameworks reveals significant overlap but some gaps and ambiguities. These differences may reflect areas where understandings of competencies are still evolving in the undergraduate biology community, warranting future research. We envision the BioSkills Guide supporting a variety of applications in undergraduate biology, including backward design of individual lessons and courses, competency assessment development, and curriculum mapping and planning.