Objective: To describe the development, evaluation, and psychometric properties of a new instrument that measures fertility preservation (FP) knowledge in women with cystic fibrosis (CF) titled the Knowledge of FP in Women With CF Instrument (KFP-WCFI). Design: The 10-item KFP-WCFI was developed and evaluated through a cross-sectional survey. Setting: Participants were recruited nationally from CF Foundation–accredited CF clinics and via snowball sampling. Participants: Fifty women with CF ages 18 through 35 years completed the instrument. Measurements: Construct validity was assessed using confirmatory factor analysis (CFA). In the CFA, the model fit was evaluated using standardized root mean square residual, root mean square error of approximation, and comparative fit index. Cronbach's alpha was used to examine internal consistency reliability. The criterion validity was assessed using inferential statistics. Results: The CFA with two subscales, General Fertility Knowledge and Transplant-Related Fertility Knowledge, demonstrated good fit, with a standardized root mean square residual of 0.07, root mean square error of approximation of 0.06, and comparative fit index of 0.97, indicating good construct validity of the instrument. This instrument demonstrated internal consistency reliability with a Cronbach's alpha of .91 for the General Fertility Knowledge subscale and .64 for the Transplant-Related Fertility Knowledge subscale. Women who reported a pregnancy scored higher than women who did not report a pregnancy (p = .02), suggesting criterion validity. Conclusion: The newly developed KFP-WCFI appears to be a valid and reliable instrument that can be used to measure self-assessed FP knowledge in women with CF.