Lim1 encodes a LIM-class homeodomain transcription factor that is essential for head and kidney development. In the developing urogenital system, Lim1 expression has been documented in the Wolffian (mesonephric) duct, the mesonephros, metanephros and fetal gonads. Using, a Lim1 lacZ knock-in allele in mice, we identified a previously unreported urogenital tissue for Lim1 expression, the epithelium of the developing Müllerian duct that gives rise to the oviduct, uterus and upper region of the vagina of the female reproductive tract. Lim1 expression in the Müllerian duct is dynamic, corresponding to its formation and differentiation in females and regression in males. Although female Lim1-null neonates had ovaries they lacked a uterus and oviducts. A novel female mouse chimera assay was developed and revealed that Lim1 is required cell autonomously for Müllerian duct epithelium formation. These studies demonstrate an essential role for Lim1 in female reproductive tract development.