Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome results from the incomplete development of the embryonic Müllerian ducts, which give rise to the female reproductive tract including oviducts, uterus, and upper third of the vagina. MRKH syndrome affects 1 in 4500 women and accounts for 10% of primary amenorrhea cases. Type I MRKH is characterized by the absence of a fully developed uterus and vagina without any associated malformations. Type II MRKH is syndromic, and features additional malformations, mostly in the renal, skeletal, and cardiovascular systems. To date, the aetiology of MRKH syndrome remains unexplained due to the lack of: 1) genome-wide approaches on large patient cohorts, and 2) functional analyses using specific mouse models. Thus, we established a recruiting program for women affected by MRKH and through their samples, we identified novel candidate genes using a combination of microarray and whole exome sequencing. Network analysis of selected variants led us to focus on Hnf1b, a transcription factor expressed in the epithelial compartment of the urogenital system, but with unknown function during Müllerian duct development. We ablated Hnf1b specifically in the epithelial cells of the Müllerian ducts by crossing a mouse line harboring a floxed Hnf1b gene (Hnf1bfl/fl) with a Wnt7aCre mouse strain. Hnf1bfl/fl;Wnt7aCre/+ mice displayed a shorter uterus and uterine hypoplasia similar to MRKH. Additionally, 15% of Hnf1b mutant mice displayed additional malformations, including unilateral kidney agenesis and skeletal abnormalities providing the first model of MRKH type II. Gene expression analysis revealed dysregulation of the Wnt pathway suggesting its importance in the development of the Müllerian ducts. In conclusion, we developed a novel strategy to identify and functionally validate genetic variants associated with MRKH syndrome. Our findings suggest that Hnf1b is critical in Müllerian duct development and a significant causative factor for MRKH syndrome.