The Pearl River Delta (PRD) is one of the most industrialized and urbanized regions in China. In order to assess the pollution status of mercury (Hg) in PRD river system, the distribution of total mercury (HgT) and methylmercury (Me-Hg) in sediment from a large river named Dongjiang (DJ) was for the first time investigated. HgT concentrations in sediment increased from the upstream to downstream of Dongjiang area and at most sites of DJ were significantly higher than the background values, which suggested that the DJ was contaminated with Hg, especially in the downstream of DJ. Me-Hg concentrations in sediment of DJ ranged from 0.56 to10.62ng/g, and were significantly correlated with HgT and organic matter. In order to determine the potential Hg contamination sources, typical sediments from different parts of DJ were chosen for Hg isotope analysis. The results showed that the mass-dependent fraction (MDF) in the sediments varied significantly (δ 202 Hg: −2.35 to−0.60‰), and the mass-independent fraction (MIF) in the sediments also varied considerably (Δ 199 Hg: −0.02 to −0.27‰). The samples with the highest HgT located in the industrial area had the highest δ 202 Hg (−1.14‰ to −0.60‰) measured values and insignificant MIF (Δ 199 Hg: −0.04 to −0.01‰). Meanwhile, the samples with the lowest HgT located in the background area had the lowest δ 202 Hg (−2.16‰ to −1.55‰) and Δ 199 Hg (−0.20 to −0.27‰) measured values. Unlike the above two cases are the samples located in the urban area, which have relative lower MDF (δ 202 Hg: −2.35‰ to −1.96‰) and small but significant MIF (Δ 199 Hg: −0.10‰ to −0.08‰) with relative higher HgT. We demonstrated that the dominant Hg sources in DJ sediments could be categorized as the regional background, urban and industrial sources. In our study, we demonstrated that Hg stable isotope method could serve as an effective tool for tracing mercury contamination sources in the environment.