Müllerian ducts are paired embryonic tubes that give rise to the female reproductive tract. In males, the Müllerian ducts regress, while in females, the ducts differentiate into the Fallopian tubes, uterus and upper vagina (in humans) and the oviduct (in birds and mice). Many human disorders of duct formation currently lack a molecular diagnosis. The most common is Mayer-Rokitansky-Kuster-Hauser syndrome, characterized by complete agenesis of the female reproductive tract. While Müllerian duct anomalies are uncommon (0.5-6.7% of the population), they are associated with infertility, endometriosis, and reproductive tract cancers, and their etiology is largely unknown. As such, studies on the genetic regulation and cellular events of Müllerian duct development are critical for shedding light on duct disorders. The chicken embryo is a useful model for duct formation, which is largely conserved between avians and mammals. This study conducted single-cell RNA sequencing of chicken Müllerian ducts to identify the different cell types responsible for duct formation, and define their origins and lineage trajectories. Single-cell analysis was conducted at key phases of duct development - specification and invagination of duct precursors in both sexes, duct elongation in both sexes, duct regression in males, and asymmetrical duct regression in females (avian-specific). Notably, this is the first reported single-cell analysis of Müllerian duct development in any species. The single-cell analysis reveals greater cell complexity in the developing Müllerian duct than previously thought, identifying two distinct sub-populations of Müllerian surface epithelium, four subpopulations of Müllerian duct mesenchyme, and three subpopulations of Müllerian duct epithelium. This study also implicated novel candidate genes in duct formation, including key regulators of the NOTCH signalling pathway such as NOTCH1, NOTCH2, JAG1 and JAG2. Lastly, this study characterizes sexual differentiation of the ducts at the cellular level, including bilateral duct regression in males and asymmetrical duct regression in females.