Multiple mouse embryonic stem (ES) cell banks expand the capability to characterize functions of genes implicated in human disease and to develop mouse models for the further understanding of disease pathology. Genetic diseases that result in hearing loss can provide insight into causative molecular mechanisms for deafness. We utilized BayGenomics, the public mouse ES cell bank, to identify gene-trapped ES cell lines associated with hearing loss. We identified two gene-trapped ES cell lines specific for the non-muscle myosin heavy chain class IIA or myosin heavy chain IX (Myh9). Inherited mutations in the Myh9 gene have been linked to non-syndromic hereditary hearing impairment DFNA17 as well as 'MYH9-related disease' characterized by macrothrombocytopenia, leukocyte inclusions, and in some patients deafness. Mutant Myh9 mice were derived from one of these ES cell lines that underwent germline transmission for in-depth otological examination. No homozygous mice however were identified at birth, consistent with recently published data describing the embryonic lethality of homozygous mutations in Myh9. We provide evidence that adult heterozygous Myh9 mouse inner ears contain half wild-type levels of Myh9 mRNA. Hearing loss however was not observed in heterozygous Myh9 mice in contrast to human Myh9-related diseases. Aged heterozygous Myh9 mice also did not show signs of cochleosaccular degeneration common in DFNA17. Although inheritance of Myh9 mutations in humans is dominant, we conclude that heterozygous loss of Myh9 is not critical to hearing function in mice by itself.