Multiplex separation of mixed cell samples is required in a variety of clinical and research applications. Herein, we present an acoustic microchip with multiple outlets and integrated pre-alignment channel to enable high performance and label-free separation of three different cell or particle fractions simultaneously at high sample throughput. By implementing a new cooling system for rigorous temperature control and minimal acoustic energy losses, we were able to operate the system isothermally and sort suspensions of 3, 5 and 7 μm beads with high efficiencies (>95.4%) and purities (>96.3%) at flow rates up to 500 μL min-1 corresponding to a throughput of ∼2.5 × 106 beads per min. Also, human viable white blood cells were successfully fractionated into lymphocytes, monocytes and granulocytes with high purities of 96.5 ± 1.6%, 71.8 ± 10.1% and 98.8 ± 0.5%, respectively, as well as high efficiencies (96.8 ± 3.3%, 66.7 ± 3.2% and 99.0 ± 0.7%) at flow rates up to 100 μL min-1 (∼100 000 cells per min). By increasing the flow rate up to 300 μL min-1 (∼300 000 cells per min) both lymphocytes and granulocytes were still recovered with high purities (92.8 ± 1.9%, 98.2 ± 1 .0%), whereas the monocyte purity decreased to 20.9 ± 10.3%. The proposed isothermal multiplex acoustophoresis platform offers efficient fractionation of complex samples in a label-free and continuous manner at thus far unreached high sample throughput rates.