The quartz vein-type gold deposits in Southeastern Guizhou, as one of the important parts of the Xuefeng Polymetallic ore belt, have showed the potential prospect for exploration. However, it still remains controversial on understanding the mineralized mechanism for this region, especially concerning the origin of metallogenetic materials, geochronology and geodynamical setting of mineralization. In this paper, arsenopyrite, one of the gold-bearing mineral, was used for Re-Os dating in two major gold deposits (Pingqiu and Jinjing). The results showed that altered slate type arsenopyrites from Pingqiu gold deposit yield an isochron age of 400±24 Ma, with an initial 187Os/188Os ratio of 1.24±0.57 (MSWD=0.96); however, large quartz vein-type arsenopyrites from Jinjing gold deposit yield an isochron age of 174±15 Ma, with an initial 187Os/188Os ratio of 8.04±0.45 (MSWD=1.07). Therefore, as sulfide Re-Os isotope dating, it is identified that there do exist at least two significant gold mineralization stages in this region. The former age belongs to Caledonian period, and is consistent with the formation age of EW trending shear zone in the region; the latter belongs to Indosinian-Yanshanian period, which may be associated with the contemporaneous tectonic-magma activities as revealed by widely exposed intermediate-acid rocks in Xuefeng area and the concealed intrusive bodies in Southeast Guizhou province. In addition, the initial 187Os/188Os ratios suggest that the metallogenic materials are derived from the crust rock.