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This paper presents the experimental results of composition changes of heavy fuel oil by simulating weathering in static seawater under natural environmental conditions. The results indicate: n-C10 to n-C15 were lost gradually in 24 weeks and the relative abundance of alkanes with long chains (n-C19) increased markedly. The aromatic compounds with less than two rings (except C4N) were completely lost in 24 weeks and CnP and CnD became the main aromatics in the heavy fuel oil after 24 weeks. The ratios of n-C17/Pristane (Pr) and n-C18/Phytane (Ph) were suitable for identifying lightly weathered (3 weeks) heavy fuel oil. The ratios of n-C17/n-C18 and Pr/Ph were suitable for identifying moderately weathered heavy fuel oil (12 weeks); the ratios of C2D/C2P and C3D/C3P did not change significantly in 24 weeks and were more suitable for identifying moderately weathered heavy fuel oil (24 weeks).