Here we report a low-pressure bead packing technique for the robust integration of high-performance chromatography columns in poly(dimethylsiloxane) microfluidic devices made by multilayer soft lithography (MSL). A novel column geometry featuring micrometer-sized bypass channels along the entire length of the separation channel is used to achieve rapid packing of multiple high-quality bead bed columns in parallel with near-perfect yield. Pulse tests show that these microfluidic columns achieve exceptional reproducibility and efficiency, with measured plate counts of 1,650,000/m ± 7%, corresponding to a reduced plate height of h = 0.12 ± 7%. The combination of high-performance chromatography columns and valve-based microfluidics offers new opportunities for the integration of sample processing with preparative and analytical separations for biology and chemistry.