In rainfed lowland rice fields, developing hardpan soil layers must be important to pool rainfall, but during drought that also restricts water movement from below it. We investigated whether the hardpan can maintain a contrast of deuterium/hydrogen isotopic composition ('D) in soil water under field condition. The experimental site was at Rajshahi in north-west Bangladesh. The hardpan soil layers had developed around 0.2 m soil-depth in the field. Soil water from either above or below the hardpan was collected non-destructively with porous cups installed into the field. Using an isotopic ratio mass spectrometer, 'D value of each water sample was determined. During the sampling period, the field surface varied from water-saturated to unsaturated status with rainfall event. While the 'D values fluctuated by the influence of rainfall, significant differences in the 'D values were always detected between above and below the hardpan, revealing heterogeneity in the 'D values persisted with the hardpan. The effect of hardpan on retention of the 'D signature was further confirmed in a laboratory experiment using intact soil columns collected from a paddy field at Nagoya University. The natural 'D signature in rainfed lowland rice field may be useful to identify certain genotypes that demonstrate in situ capability of water acquisition from below the hardpan through 'D analysis of the xylem sap.