The upper mantle section of the Cuobuzha ophiolite in the northern subbelt of the Yarlung Zangbo suture zone in southwest Tibet comprises mainly clinopyroxene (cpx)-rich and depleted harzburgites. Spinels in the cpx-harzburgites show lower Cr# values (12.6–15.1) than the spinels in the harzburgites (26.1–34.5), and the cpx-harzburgites display higher heavy rare earth element concentrations than the depleted harzburgites. The harzburgites have subchondritic Os isotopic compositions (0.11624–0.11699), whereas the cpx-harzburgites have suprachondritic 187Os/188Os ratios (0.12831–0.13125) with higher Re concentrations (0.380–0.575 ppb). Although these geochemical and isotopic signatures suggest that both peridotite types in the ophiolite represent mid-oceanic ridge–type upper mantle units, their melt evolution trends reflect different mantle processes. The cpx-harzburgites formed from low-degree partial melting of a primitive mantle source, and they were subsequently modified by melt-rock interactions in a mid-oceanic ridge environment. The depleted harzburgites, however, were produced by remelting of the cpx-harzburgites, which later interacted with mid-oceanic ridge basalt– or island-arc tholeiite–like melts, possibly in a trench–distal backarc spreading center. Our new isotopic and geochemical data from the Cuobuzha peridotites confirm that the Neo-Tethyan upper mantle had highly heterogeneous Os isotopic compositions as a result of multiple melt production and melt extraction events during its seafloor spreading evolution.