AbstractBackground: Tbx2, Tbx3, Tbx4, and Tbx5, members of the Tbx2 subfamily of T‐box transcription factor genes, are important for many aspects of embryonic development and mutations in some human TBX2 subfamily genes cause developmental syndromes. In addition, TBX2 and TBX3 are overexpressed in a variety of cancers, including reproductive system cancers. This study characterizes the expression of Tbx2 subfamily genes during development of the reproductive system. Results: We show that these genes are expressed in both the internal and external reproductive systems. Tbx2 is expressed in gonads and genital ducts, the Wolffian and Müllerian ducts, while Tbx3 is only expressed in genital ducts. Tbx4 is expressed in embryonic and postnatal germ cells. All four genes are expressed in mesenchyme in external genitalia, with Tbx3 and Tbx5 expression in the epithelium as well. Conclusion: This study lays the foundation for investigation of functional requirements for Tbx2 subfamily genes in development of the mammalian reproductive system. Developmental Dynamics 241:365–375, 2012. © 2011 Wiley Periodicals, Inc.