The presence of coal-based power plants and coal mine in the Waterberg area subjects the Mokolo River to potentially toxic elements (PTEs) such as mercury (Hg). Mercury is an extremely toxic element. Thus, monitoring and chemical speciation of Hg in water bodies; particularly in sediments is a vital tool for assessing water quality. The objective of this study was to investigate the levels of Hg(II) and methyl Hg (MeHg(I)) in sediment samples collected from Mokolo River in different seasons, as well as examining factors such as pH, temperature and organic matter content, which could affect Hg methylation rates. An ultrasonic based method was used for the extraction of Hg species in sediments. This was followed by the chromatographic separation and detection of Hg(II) and MeHg(I) by the on-line coupling of high-performance liquid chromatography (HPLC) to inductively coupled plasma-mass spectrometry (ICP-MS). A solution containing HCl and 2-mercaptoethanol was employed for the extraction of Hg species in sediments. Separation of the two species of Hg was achieved using isocratic elution mode with a mobile phase containing L-cysteine, 2-mercaptoethanol, ammonium acetate and methanol. The accuracy of the method was checked and yielded a percentage recovery of 86%. The Hg(II) concentrations ranged from 38.4 to 89.05 ng g−1 and 34.8 to 57.3 ng g−1 in low and high flow seasons, respectively. The concentrations of MeHg(I) ranged from 0.702 to 4.5 ng g−1 and 0.5 to 2.5 ng g−1 in the low and high flow seasons, respectively. Factors such as pH and temperature were found to influence the methylation rates, however correlation couldn’t be established to organic matter content due to similar amount of organic matter in all the samples.