Double focus fluorescence correlation spectroscopy (dfFCS) was used to determine electrophoretic mobilities of short double-stranded DNA (dsDNA)-fragments (75 base pairs (bp) -1019 bp) in microfluidic channels. The electrokinetic flow profile across a microchannel was measured with 1 mu m spatial resolution and separated in electroosmotic and electrophoretic contributions. Experiments show that the free solution mobility is independent of DNA length. The diffusion constant is additionally determined by FCS and follows a length dependent rod-diffusion model. We interpret the electrophoretic mobilities using a modified Nernst Einstein relation, which additionally takes Manning condensation and counterion induced hydrodynamic retardation forces into account. In 3% w/v polyethylene oxide (PEO)-network (M-r 3 (.) 10(5) Dalton) the electrophoretic velocities become size-dependent with a power-law exponent between 0.28 and 0.31. Mixtures of dsDNA-fragments exhibit distinguishable peaks in the dfFCS cross-correlation function. The potential of dfFCS for realtime micro-analysis in terms of speed and spatial resolution is discussed.