•Fundamental spatio-temporal changes of wetlands took place in the lowland areas of the Czech Republic.•Wetlands mostly disappeared in favour of arable land due to extensive agricultural management.•Results help to understand historical dynamics of wetlands at the landscape level.•Methodology helps to determine continuous biotopes and locations for wetland restoration. Wetland biotopes play many roles in the landscape. The primary objective of this study was to analyse historical long-term changes of wetlands in the lowland areas of the Czech Republic, with emphasis on their spatio-temporal changes in the landscape and with stress on the use of different types of information sources. The following research questions were asked: (1) What are the wetland spatio-temporal changes (expressed through the number and size of continuously existing, extinct, and recent wetlands) and wetland change trajectories (expressed by quantifying and localising LULC categories that replaced extinct wetlands as well as categories of LULC at the expense of which wetlands occur) in the lowland landscape of the Czech Republic? (2) What methodological conclusions and recommendations for the investigation of wetland changes in a landscape can be derived from the study? The sources used for the study included historical maps from the Stable Land Registry 1841, a current orthophoto map (2015), the DIBAVOD database, LPIS Land-Parcel Identification System database verification, the Czech Land Registry, and field mapping (2015). Three wetland biotope types were examined: swamps and marshes, waterlogged meadows, and waterlogged woody vegetation. Spatial analysis using GIS tools (intersection, symmetrical difference) was performed to analyse the spatio-temporal wetland changes. The total wetland area has decreased dramatically from 5762ha (over 9.5% of the territory included in the study) in 1825–1843 to 54ha (0.9%) in 2014. Extinct wetlands predominate (99.1%) in the area, whilst recent and continuous wetlands are nearly identical (0.5% and 0.4%, respectively). A selected sample of 10 cadastral districts was used to examine the error against the DIBAVOD method and find that the error is relatively low (0.13%). The results of this study document fundamental spatio-temporal changes (expressed through change trajectories) of wetland biotopes at the landscape level in the lowland and hilly areas of the Czech Republic. The most frequent change type is wetland loss in favour of arable land (nearly one-half of all wetlands) due to extensive agricultural management. The use of the DIBAVOD method is effective, taking the relatively low error into account. The results of the study substantially contribute to the development of methods for the investigation of spatio-temporal changes in wetlands and other selected biotopes in the context of the landscape.