The Jiadi gold deposit is one of the typical gold deposits in the basalt distribution area of southwestern Guizhou. The Au-bearing pyrite in this area is generally zoned and has the characteristics of multi-period and multi-source, therefore, it is very challenging to delineate the source of ore-forming materials and the genesis of the deposit. Based on the field observation and laboratory microscope study, three stages of pyrite were recognized. The pre-ore pyrite is strawberry-shaped pyrite, which is typically formed in sedimentary period. The ore stage pyrite can further be subdivided into zoned and non-zoned types. The zoned pyrite usually occurs as veins, euhedral-subhedral, 10 to 100 μm in size, the zonation around the pyrite cores is obvious in back-scattered electron images, and the cores are highly fissured. The non-zoned pyrite is commonly pentagonal-dodecahedral pyrite of about 50 μm in size. The post-ore pyrite is large, cubic or irregular in shape, and zonation is common. LA-MC-ICP-MS in situ sulfur isotope analysis of pyrite of different stages showed that the δ34S values of the pre-ore pyrite vary from −23.47% to −19.76%, indicative of sedimentary S. δ34S values of the ore-stage pyrite are −1.39% to 4.45%, and δ34S values of the post-ore pyrite are −1.57% to −0.91%, suggestive of magmatic S. On the basis of previous studies, it is inferred that the ore-forming materials were mainly derived from the mantle, along with some crustal materials. The ore mineralization may involve two processes: firstly, the reaction between ankerite and a CO2-rich fluid releases Fe2+, Mg2+, Ca2+ ions, forms dolomite and necessary Fe for the subsequent sulfidation or pyritization. The H3AsO30, [Au(HS)2]− and H2S-rich reductive fluid reacts with Fe2+ to form arsenian pyrite and pyrite, during which gold enters into the lattice of arsenian pyrite in the form of Au+ and consequently, the gold enrichment. © 2023 Science Press. All rights reserved.