Acoustophoresis in microfluidic structures has primarily been reported in silicon microfabricated devices. This paper demonstrates, for the first time, acoustophoresis performed in isotropically etched glass chips providing a performance that matches that of the corresponding silicon microdevices. The resonance mode characteristics of the glass chip were equal to those of the silicon chip at its fundamental resonance. At higher order resonance modes the glass chip displays resonances at lower frequencies than the silicon chip. The cross-sectional profiles of acoustically focused particle streams are also reported for the first time, displaying particles confined in a vertical band in the channel center for both glass and silicon chips. A particle extraction efficiency of 98% at flow rates up to 200 microL/min (2% particle concentration) is reported for the glass chip at the fundamental resonance. The glass and silicon chips displayed equal particle extraction performance when tested for increasing particle concentrations of 2-15%, at a flow velocity of 12.9 cm/s for the glass chip and 14.8 cm/s for the silicon chip.