Occult hepatitis B virus (HBV) infection (OBI) is defined as the presence of HBV DNA in the liver with or without detectable HBV DNA in the serum of individuals testing HBV surface antigen (HBsAg) negative using currently available assays. The prevalence of OBI among patients receiving hemodialysis (HD) treatment remains poorly characterized in South Africa despite the high prevalence of HBV. We sought to determine the prevalence of OBI in HD units in tertiary hospitals of KwaZulu‐Natal and to characterize the HBV S gene mutations potentially responsible for OBI. A cross‐sectional descriptive study of residual diagnostic plasma samples from 85 HBsAg‐negative patients receiving HD treatment was included. The PreS/S gene was amplified with a nested HBV polymerase chain reaction for downstream next‐generation sequencing, to determine the viral genotype and identify S gene mutations associated with OBI. Nine of the 85 samples had OBI, based on detectable HBV DNA. The point prevalence of OBI was 10.6% (95% control interval: 5.5%‐19.1%). Phylogenetic analysis of the samples with OBI showed that all belonged to genotype A. Three (~33%) samples had mutations in the major hydrophilic region (MHR) within the S gene, three (~33%) had mutations within the S gene but outside the MHR, whilst the remaining three had no mutations observed. The prevalence of OBI in HBsAg‐negative patients undergoing HD was 10.6%, suggesting that OBI is a clinically significant problem in patients with HD in this region. The screening methods for HBV infection need to be revised to include nucleic acid testing.