The upper mantle section of the Dargai ophiolite along the Indus Suture Zone, also known as the Main Mantle Thrust of northern Pakistan, consists mainly of clinopyroxene-bearing (Cpx)-harzburgites, depleted harzburgites, and dunites. Here we document a two-stage origin for these ultramafic rocks using new mineral and whole-rock geochemistry, integrated with Re-Os isotopic data of these upper mantle peridotites. Chromian spinel in the Cpx-harzburgites have significantly lower Cr# values (15.2-20.7) than the depleted harzburgites (38.4-75.2) and dunites (75.0-81.9). The Cpx-harzburgites are characterized by high average Al2O3 (2.04 wt%), CaO (2.26 wt%), sigma(REE) (24.3 ppb), and Os-187/Os-188 values ranging between 0.12495 and 0.12997. In contrast, the depleted harzburgites and dunites are characterized by lower average Al2O3 (0.51 wt% and 0.14 wt%, respectively), CaO (0.68 wt% and 0.19 wt%, respectively), and sigma(REE) (3.41 ppb and 0.92 ppb, respectively). The Os-187/Os-188 of the depleted harzburgites range between 0.11699 and 0.12612, values that are less radiogenic than those of the Cpx-harzburgites. The mantle-normalized trace element patterns of the Cpx-harzburgites reflect low degrees of partial melting (similar to 10-15%), whereas those of the depleted harzburgites and dunites record somewhat higher degrees of partial melting (similar to 15-23% additional melting of the Cpx-harzburgites). The low degree of melting, coupled with low degrees of melt extraction, therefore point to a spreading center at amid-ocean ridge (MOR) or in a distal forearc basin. In contrast, the depleted harzburgites and dunites formed during the second stage of melting and related refertilization, and were clearly linked to a supra-subduction zone (SSZ) setting. The Re-Os isotopic compositions of the Dargai Complex peridotites provide model ages of ca. 250 and ca. 450 Ma recording pro-tracted tectonic events in close association with the geodynamic evolution of the Neo-Tethyan, Rheic, and Proto-Tethyan oceans. The extremely heterogeneous Os isotopic compositions of this part of the Neo-Tethyan upper mantle reflect multiple melt production and melt extraction processes throughout its tectonic evolution.