Microplastic pollution in global aquatic environments has aroused increasing concern in recent years. In this study, the occurrence of microplastics in multiple environmental compartments was investigated in Poyang Lake, the largest freshwater lake of China. The abundance of microplastics was respectively 5–34 items/L for surface waters, 54–506 items/kg for sediments, and 0–18 items per individual for wild crucians (Carassius auratus). The distribution of microplastics in Poyang Lake varied heterogeneously in space, with the highest abundance being observed in the middle region of the lake for surface waters and in the northern region for sediments. Anthropogenic and topographic factors were speculated to be the major factors affecting the abundance and distribution of microplastics. The majority of the detected microplastics were found with a size of < 0.5 mm, with fibrous and coloured being the predominant characteristics. Polypropylene (PP) and polyethylene (PE) were the major polymer types of the selected plastic particles, indicating that domestic sewage and fishing activities might be the main sources of microplastics in the lake. No significant correlation was observed between microplastic abundance in surface water and sediment samples. Our results demonstrated the wide occurrence of microplastics in water, sediment and biota of the Poyang Lake, which may assist in extending our knowledge regarding microplastics pollution in inland freshwater systems.