Non-syndromic hearing loss (NSHL) is a hereditary disorder that affects many populations. Many genes are involved in NSHL and the mutational load of these genes often differs among ethnic groups. Claudin-14 (CLDN14), a tight junction protein, is known to be associated with NSHL in many populations. In this study, we aimed to identify the responsible variants in 3 different Yemeni families affected with NSHL. Firstly, clinical exome sequencing (CES) performed for 3 affected patients from these different families identified a new nonsense variant (c.414G > A) in CLDN14. This variant was then confirmed by Sanger sequencing and PCR-RFLP. Subsequently, four microsatellite markers were used to genotype these families, which revealed a founder effect for this variant. Overall, this study illustrates the implication of the CLDN14 gene in the Yemeni population with NSHL and identifies a new founder variant.