The Wunuer Pb–Zn–Ag–Mo deposit is a newly explored polymetallic ore deposit located in the middle segment of the Great Xing'an Range, Inner Mongolia, NE China. Three stages of mineralization, composed of an early porphyry stage, an intermediate hydrothermal (cryptoexplosive breccia) stage, and a later epithermal stage, have been identified in the Wunuer deposit. Sphalerite is one of the principal metal sulphides in both hydrothermal and epithermal stages, and thus two generations of sphalerite, with the first‐generation sphalerite (Sp1) precipitated in hydrothermal stage and the second‐generation sphalerite (Sp2) precipitated in epithermal stage, were discriminated. The Sp1 is generally euhedral, transparent and colour‐zoned, and is usually replaced by Sp2, galena and chalcopyrite. The Sp2 is generally anhedral, opaque and black in colour. Chalcopyrite inclusions in Sp1 and Sp2 have different genetic mechanisms: chalcopyrite inclusions in Sp1 were produced by replacement as the result of interaction of sphalerite with Cu‐rich fluids, while chalcopyrite inclusions in Sp2 were produced by coprecipitation of sphalerite and chalcopyrite during crystal growth. Electron probe microscope analyser and laser ablation inductively coupled plasma mass spectrometry in‐situ analysis techniques have been used to obtain chemical compositions of sphalerite. The Sp1 is enriched in Cd and In, and depleted in Mn, Fe, Cu, Ag, Sb and Pb. In contrast, the Sp2 is enriched in Mn, Fe, Cu, Ag, Sb and Pb, and depleted in Cd and In. Elements of Pb, Ag, Bi and some of Fe, Cu concentrated in Sp2 are greatly attributed to micro‐mineral inclusions like chalcopyrite and galena, while most elements concentrated into Sp1 are generally incorporated into crystal structure in mechanisms of direct substitution (e.g. Fe2+ → Zn2+) or coupled substitution ((Cu+ + In3+) → 2Zn2+). In‐situ sulphur isotope results reveal similar slightly positive sulphur isotope compositions (+1.55‰ to +3.33‰ δ34SV‐CDT for Sp1, and +1.71‰ to +2.34‰ δ34SV‐CDT for Sp2) of the two generations of sphalerite, implying a magmatic material for both Sp1 and Sp2. Fluid inclusions in molybdenite‐bearing quartz vein, Sp1 and Sp2‐coexisting quartz have been studied to reveal fluid temperature and salinity evolution from porphyry to hydrothermal and to epithermal stage mineralization. Primary fluid inclusions in porphyry stage quartz homogenized to a liquid phase by high homogenization temperatures (range from 385 to 416°C, with an average of 399°C), with salinities ranging from 12.4 to 25.3 wt% (average of 19.4 wt%). The Sp1 was precipitated from fluids with medium temperature (302–317°C) and medium salinity (5.1–7.3 wt% NaCl) in the hydrothermal stage, and later been altered by metasomatic fluids with low temperature (142–186°C) and low salinity (0.7–5.3 wt% NaCl). The Sp2 was precipitated from fluids with low temperature (150–234°C) and low salinity (0.4–6 wt% NaCl) in the epithermal stage.