Increases in positive end-expiratory pressure (PEEP) improve arterial oxygenation in preterm infants, but the effects on cardiopulmonary haemodynamics are understood poorly. We aimed to determine the effect of increased PEEP on cardiopulmonary haemodynamics and to compare measurements from indwelling flow-probes with Doppler echocardiography. Preterm lambs (129 ± 1d) were ventilated initially with a tidal volume (V_T) of 7 mL/kg and 4 cmH₂O of PEEP. In Ramp lambs (n=7), PEEP was increased by 2 cmH₂O increments to 10 cmH₂O and then in decrements back to 4 cmH₂O. PEEP was unchanged in Controls (n=6). Doppler echocardiographic flow measurements in the left pulmonary artery (LPA) and ductus arteriosus (DA) were correlated with flow-probe measurements. Compared to Controls, high PEEP reduced LPA flow from baseline (10 cmH₂O PEEP: 43 ± 8 % vs. Control: 83 ± 21 %; p=0.029). High PEEP increased the proportion of right-to-left shunting through the ductus arteriosus (DA), with a trend to an increased Oxygenation Index (OI) compared to controls (OI: 44.5 ± 13.5 at 10 cmH₂O PEEP vs. 19.4 ± 4.5 in Controls; p=0.07). Increasing PEEP decreased heart rate (17 beats/min; p=0.03) and tended to lower systolic arterial pressure (5.0 mmHg; p=0.052) compared to Controls. Doppler echocardiography measurement of LPA flows correlated strongly with indwelling flow-probe (r²=0.73, p<0.001), except during highly turbulent flows. Increases in PEEP have significant cardiopulmonary consequences in preterm lambs, including reduced LPA flow and increased R-L shunt through the DA. These changes are likely due to the concomitant increase in downstream pulmonary vascular resistance and increased cardiovascular constraint induced by PEEP.