Introduction: Cord blood (CB) is rich in hematopoietic stem cells. There has been significant growth in cord blood banks (CBBs) throughout the world. In Malaysia, the CB units cryopreserved in public CBB is below its optimal level. Lack of knowledge about CB banking among mothers in other countries influenced their decision to donate CB, however, there is no local data to support this in Malaysia. Moreover, no validated and reliable tool is available for measuring local women’s knowledge about CB banking. In this study, a questionnaire to assess pregnant women’s knowledge about CB banking was developed and validated. Method: Several steps were taken to develop the knowledge items in the questionnaire which includes comprehensive literature review, content validity by a panel of experts, and face validity by a group of pregnant women. The questionnaire was developed in Malay language and contained 18 items. After modifications, the self-administered questionnaire was distributed to 121 pregnant women to assess its psychometric properties using two-parameter logistic item response theory analysis and internal consistency reliability analysis. Results: The majority of the knowledge items showed acceptable difficulty and discriminatory values. The Cronbach’s alpha and ICC values were 0.831 and 0.887, respectively, indicating good reliability. All 18 knowledge items were retained for the final version of the questionnaire. Conclusion: The newly developed questionnaire demonstrated acceptable psychometric properties and can be used as a reliable tool to assess knowledge about CB banking among pregnant women in the local population.