Garnet pyroxenites, spinel pyroxenites, and eclogites in the Moldanubian Zone of the Bohemian Massif form layers and lenses in mantle-derived peridotites that are enclosed by migmatitic gneisses and granulites. We have analysed major and trace elements, and Sr, Nd and oxygen isotopes for a suite of pyroxenites, which vary in composition and origin, from nine localities (Bečváry, Horní Bory, Drahonín, Níhov, Mohelno, Nové Dvory, Horní Kounice, Karlstetten and Meidling-im-Tal) in the Gföhl Unit and Kutná Hora Complex. Based on conventional geothermobarometry, most pyroxenites yield a restricted range of temperatures (∼875–975 °C) over a wide span of pressures (∼1·0–3·0 GPa). The pyroxenite suite exhibits large variations in elemental and isotopic compositions, reflecting its complex origin and evolution. Based on the rare earth element (REE) compositions of clinopyroxene (Cpx), three types of pyroxenite can be distinguished: Type A with a light REE (LREE)-depleted patterns, Type B with an LREE-enriched pattern and Type C with a convex-upward REE pattern. Such REE patterns reflect derivation of the melts from depleted (Type A) and enriched (Types B and C) mantle sources. Pyroxenites from eight localities originated as high-pressure crystal cumulates from transient basaltic melts migrating through the lithospheric mantle. In contrast, pyroxenites at the Bečváry locality represent fragments of metamorphosed gabbroic cumulates from oceanic crust. For the pyroxenite suite as a whole, a positive correlation between Sr/Nd and Eu/Eu*, radiogenic 87Sr/86Sr and negative εNd values in clinopyroxene, and variable δ18O values in coexisting garnet argue for the presence of a crustal component in the parental pyroxenite melts. Variations in compatible elements (Ni, Sc, and Co) indicate that combined assimilation and fractional crystallization was important in the evolution of most of the pyroxenite parental melts, although fractional crystallization alone is recorded by the fragments of oceanic crust, perhaps reflecting their pre-subduction crystallization history.