The halogen Cl is one of the sensitive indicators for the origin of ore-forming fluids. However, accurate analysis of trace Cl in minerals or glasses remains a challenge. Here, we establish an electron probe microanalysis (EPMA) method for determination of Cl in sphalerite by investigating the key parameters such as accelerating voltage, analytical crystals, beam current, peak counting time, primary calibration standards, and secondary fluorescence effects. Detection limit (3σ) for Cl can be lowered to 14 ppm. The proposed method was used to analyze Cl and other minor/trace elements such as Fe and Ge in sphalerite from the Shanshulin Mississippi Valley type Zn-Pb deposit in the southwestern China. EPMA mapping and profile analyses show that Cl is heterogenous (from below detection limit to 869 ppm) in sphalerite with zoning patterns. Random point analyses show the highest Cl content in the studied sphalerite is 2730 ppm. No correlation of Cl with sphalerite color, Fe, Ge, S, and Zn indicates that Cl behaves differently from other elements. Considering detectable and various Cl contents in sphalerite at the grain scale, between samples, and between deposits, Cl together with other elements can be used to constrain the ore genesis.