The present invention provides compositions and methods for the detection and characterization of mutations associated with non-syndromic hearing impairment. More particularly, the present invention provides compositions, methods and kits for using invasive cleavage structure assays (e.g. the INVADER assay) to screen nucleic acid samples, e.g., from patients, for the presence of any one of a collection of mutations in the Connexin 26, or gap junction beta 2, gene associated with non-syndromic hearing loss.