Abstract Mutations in miRNA genes have been implicated in hearing loss in human families and mice. It is also possible that mutations in miRNA binding sites of inner ear targets alter gene expression levels and lead to hearing loss. To investigate these possibilities we screened predicted target genes of the miR‐183 miRNA cluster known to be expressed in the inner ear sensory epithelium. In one Iranian family segregating autosomal recessive non‐syndromic hearing loss (ARNSHL), we identified a homozygous variant in a predicted miR‐96/182 binding site in the 3′UTR of the RDX (DFNB24) gene. However, in vitro functional studies showed that this site is not a functional target for miR‐96/182. We extended our study to include the miR‐183 genes themselves and 24 additional predicted target genes of the miRNA‐183 cluster. Screening these miRNAs and target sequences in numerous families segregating either autosomal dominant non‐syndromic deafness (ADNSHL) or ARNSHL did not identify any potential deafness‐causing mutations. These results suggest that mutations disrupting gene regulation by the miR‐183 cluster are not a common cause of human hearing loss. © 2010 Wiley‐Liss, Inc.