Boninites in many supra-subduction zone ophiolites are thought to mark the early stage of subduction. The Troodos ophiolite in Cyprus contains a variety of volcanic rocks including the lower pillow lavas and the boninite-bearing upper pillow lavas (UPL). Here we present major, trace element and Pb isotope data for melt inclusions in olivine from the UPL on the northern flank of the Troodos ophiolite. Melt inclusions hosted by high-Mg (Fo = 89.4–93.5) olivine grains have typical high-Ca boninitic compositions with high MgO (11.2–18.4 wt%) and SiO2 (51.2–55.0 wt%) and low TiO2 (0.2–0.6 wt%) contents, in line with the existence of boninites in the northern part of the ophiolite. The melt inclusions have trace element compositions similar to the Troodos boninitic lavas and can be regarded as representing the parental magma of the boninites. The boninitic magmas were derived from high-degrees of partial melting of a refractory source at 1400 °C and 1.5 GPa based on thermobarometers and phase equilibria. The melt inclusions have significantly variable Pb isotopic compositions (208Pb/206Pb = 2.059–2.122; 207Pb/206Pb = 0.828–0.862) and are enriched in fluid-mobile trace elements (e.g., U, Sr, and Pb), indicating that the refractory source was metasomatized by slab fluids. In comparison, recalculation of literature data suggests that the lower pillow lavas were derived from a less refractory and shallower source. We propose a subduction initiation model in which melting of two unique sources was needed to form the chemostratigraphy of lavas in the Troodos ophiolite.