In order to thoroughly evaluate the genesis of orogenic gold mineralization in the context of plate tectonics, absolute age data are required to link mineralization with lithological evolution. We show that pyrite, associated with orogenic gold mineralization from the Con deposit in the ca. 2700 Ma Yellowknife greenstone belt, has an Re-Os isochron age of 2591 ± 37 Ma. This is identical to the previously proposed timing of ca. 2592 Ma for S-type pluton-related lode-gold within ca. 2660 Ma graywacke-mudstone turbidites east of the greenstone belt. The Re-Os pyrite data indicate that orogenic gold mineralization was deposited ca. 60 to 100 m.y. after the formation of the host rocks in the Yellowknife district and, when combined with the regional geologic framework, indicate that the gold mineralization formed in a distinct geodynamic setting from that during host-rock formation. Initial Os values in the pyrite (Osi = 0.78 ± 0.17) indicate a significant crustal component of Os to the ore system, compatible with metals being derived from the older host rocks. Regionally preserved granulites, migmatites, and charnockites derived, in part, from the older supracrustal sequences, formed at 2595 to 2585 Ma, corresponding to the timing of gold mineralization. S-type plutons with ca. 2592 Ma crystallization ages in the upper crust reflect this high-temperature metamorphic event; these plutons have turbidite-hosted gold deposits associated with their metamorphic aureoles. Collectively, the isotopic and regional geologic data indicate that gold mineralization was related to regional-scale crustal anatexis, which likely resulted from crustal accretion or collision outside what is now preserved of the craton.