The Yangtze River is the longest river in China; nearly one-third of the national population lives along the river. Air quality over the Yangtze River is important as it may have significant influences on the aquatic ecosystem, the health of everyone living along the Yangtze River, and regional climate change. Chemical compositions of ambient aerosol were determined during a comprehensive cruise campaign carried out along the mid–lower reaches of the Yangtze River (MLYR) in winter of 2015. The total average concentration of PM2.5 was 119.29±33.67 µg m−3, and the dominant ionic composition in PM2.5 was SO2−4 with an average concentration of 15.21±6.69 µg m−3, followed by NO−3 (13.76±4.99 µg m−3), NH+4 (9.38±4.35 µg m−3), and Ca2+ (2.23±1.24 µg m−3) in this cruise. Based on the filter samples, the concentration and chemical composition of PM2.5 were remarkably varied or fluctuated from coastal areas to inland over the MLYR region. Crustal elements (Ca, Mg, Al, and K) from floating dust showed peak concentrations in the Yangtze River Delta (YRD) region, while secondary inorganic species (SO2−4, NO−3, and NH+4) and some of the most enriched elements (Pb, As, Se, and Cd) presented high levels in central China (Wuhan region). The significant correlation between Se and SO2−4 suggested that coal combustion may play an important role in secondary inorganic aerosol formation. The relatively high enrichment factors (EFs) of Ca (EFs >100) suggested the crustal elements may derive from anthropogenic sources. Furthermore, the concentration of levoglucosan in PM2.5 and the CO column level from satellite observation were greatly enhanced in the rural areas (Anhui and Jiangxi), indicating that biomass burning may make a remarkable contribution to rural areas. The concentrations of typical tracer for heavy oil (V and Ni) significantly increased in the Shanghai port, which was mainly ascribed to ship emissions, based on the air mass source analysis and the relatively high ratio of V ∕ Ni as well. The results shown herein portray a good picture of air pollution along the Yangtze River.