Purpose: To develop and psychometrically test a comprehensive measure of preconception health knowledge. Design: Cross-sectional survey, in May and June, 2019. Setting: Alberta, Ontario, and Québec, Canada. Sample: One thousand seven hundred seventy-seven women and men with ≥1 children born in the last 5 years or planning a pregnancy in the next 5 years. Measures: Using prior literature and input from public health nurses and physicians, the Preconception Health Knowledge Questionnaire (PHKQ) was developed and comprised 25 multiple choice questions on reproductive history, sexual health, infectious diseases, chronic medical conditions, mental health, medications, immunizations, lifestyle behaviors, psychosocial stressors, and environmental exposures. Analysis: Psychometric testing was undertaken to evaluate item difficulty, discrimination, quality of response alternatives, internal consistency, and construct validity. Results: Participants had a mean total score of 15.8/25 (SD = 3.9); women and men had mean total scores of 16.2 (SD = 3.6) and 13.8 (SD = 4.7), respectively. Most items were neither too difficult nor too easy, discriminated well between participants with high and low knowledge, and had appropriate response alternatives. High internal consistency (KR-20 = 0.87) and construct validity, shown via significant correlations with education level and previous preconception care receipt, were demonstrated. Conclusion: The PHKQ is a reliable and valid tool for measuring preconception health knowledge and may be useful in identification of high-risk groups in need of preconception health education and evaluation of preconception health interventions.