River flooding is important for the ecological functioning of river floodplains. It is implicitly assumed that in many river floodplains during floods, river water is spreading all over the floodplain. We hypothesize that during flood events a spatial distribution of water types exists, which is correlated to different water sources (river water, atmospheric water and groundwater) and to the spatial distribution of vegetation types. The objective of this paper is to assess a new methodology to determine the extent of flooding and the spatial distribution of different water sources during the flood, using GPS, multispectral remote sensing and hydrochemical analyses. This methodology is applied to the Biebrza River Lower Basin, which has little human impact. Remote sensing resulted in a map distinguishing inundated areas from dry areas, which showed 85% agreement with GPS field measurements. Principal Component Analyses and Cluster Analyses on the measured water chemistry identified different water sources during the flood (river water, groundwater, rainwater) and showed the effects of human impact on water quality. River flood water dominated the entire inundation zone in the northern Lower Basin, which is narrower and steeper than the southern Lower Basin where groundwater and rainwater were significant contributors to the major part of the inundated area. Vegetation in the river flood zone is distinctly different from the rest of the floodplain. Due to mixing of ground- and rainwater, correlation analyses between vegetation and water type were not possible outside the river flood zone. The new methodology is effective in distinguishing inundated areas from dry regions and in separating river flood water from other water sources during a flood.