The Odd‐skipped related 1 (Osr1) gene encodes a zinc finger protein homologous to the Drosophila Odd‐skipped transcription factor. During mouse embryogenesis, Osr1 is expressed in multiple tissues, including the developing heart, kidney, limb, lung, and craniofacial structures. Although characterization of targeted mutant mice has revealed essential roles for Osr1 in heart and kidney development, the embryonic lethality of the Osr1 null mice has hindered investigation of its role in many other developmental processes. We report here the generation of conditional mutant mice containing two loxP sites flanking exon 2 of the Osr1 gene. Mice homozygous for this targeted Osr1 ^fneo allele are normal and fertile. Crossing the Osr1^fneo/fneo mice with the EIIa‐Cre transgenic mice resulted in Cre‐mediated deletion of the loxP‐flanked Exon2 in the germ line and mice homozygous for this deletion recapitulated the Osr1 null mutant phenotypes. The Osr1 ^fneo conditional mice will be valuable for tissue‐specific analysis of the roles of Osr1 in embryonic and postnatal developmental processes. genesis 49:419–422, 2011. © 2011 Wiley‐Liss, Inc.