Analysis of abnormal phenotypes produced by null and hypomorphic mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are hypomorphic and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6tm1Ued (Pax6fl) allele, which has a retained neo cassette, is hypomorphic for any eye phenotypes. Homozygous Pax6fl/fl and heterozygous Pax6fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak hypomorphic phenotypes, we produced compound heterozygotes with the Pax6Sey-Neu (Pax6-) null allele. Pax6fl/- compound heterozygotes had more severe eye abnormalities than Pax6+/- heterozygotes. This implies that Pax6fl differs from the wild-type Pax6+ allele, so is hypomorphic at the phenotypic level. Immunohistochemistry showed that the Pax6fl/- corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6fl allele, with a weakly hypomorphic eye phenotype, provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic hypomorphic phenotypes of floxed alleles.