Hearing loss is one of the most common sensorineural defects in humans. Autosomal-recessive nonsyndromic hearing loss (ARNSHL) is the most frequent form among inherited forms of deafness and accounts for greater than 70% of the cases. Due to extreme genetic heterogeneity of ARNSHL, many known loci have to be screened to find linkage to deafness genes or before proceeding to a genome wide analysis to identify a new locus for the disorder. Microsatellite based homozygosity mapping is an excellent option but throughput is low as it yields genotype information at only one locus per reaction. This makes screening a large number of loci very laborious and expensive. Here we describe a protocol to reduce the time and costs of microsatellite based screening. It involves selecting microsatellite markers close to the known deafness genes thereby decreasing the number of markers required to screen for each locus and multiplexing the PCR reactions. Furthermore, primers for some known microsatellites were redesigned for multiplexing and finally a protocol of genotyping with fluorescently labeled universal M13 primers was incorporated in the strategy.