The separation of target objects conjugated with magnetic particles is a significant application in biomedicine and clinical diagnosis. Conventional magnetophoresis-based devices use a sheath flow to pre-focus the particles into a single stream and typically operate at a low flow rate. We demonstrate in this work a high-throughput, sheathless, magnetophoretic separation of magnetic and non-magnetic beads in a groove-based channel, and also report on an interesting phenomenon where the same magnetic beads in the same microchannel, but with different setups, has a different particle tracing; a binary mixture of magnetic and non-magnetic beads in a diluted ferrofluid, is then fed into the channel. These magnetic beads are focused near the centreline of the channel by exploiting positive magnetophoresis and microvortices generated by grooves, whereas the non-magnetic beads are focused along the sidewalls of the channel by negative magnetophoresis and hydrophoresis. These magnetic and non-magnetic beads are separated in a wide range of flow rates (up to 80 μl min−1).