In the European Union, the socio-ecological systems underlying the maintenance of low-intensity farming systems supporting the occurrence of several species and habitats are known as High Nature Value farmlands (HNVf). Detecting trends of change in the extent and location of HNVf is essential to monitor the impact of policies on biodiversity. However, assessing changes in HNVf extent is challenging, due to the lack of tested approaches and lack of data with adequate spatial and temporal resolutions. We address such challenge by evaluating the usefulness of an existing methodological framework to analyse changes in the extent of HNVf in the agrarian region of Entre-Douro-e-Minho, Northwestern Portugal between 1989 and 2009. Changes in the extent of HNVf between 1989 and 2009 were analysed for whole study area, and within and outside areas designated for conservation. Results depicted a trend of decreasing extent of HNVf between 1989 and 2009, irrespective of being inside or outside a nature conservation designation. This provides an early warning that nature conservation designation does not ensure HNVf persistence. We consider that this research represents an advance in the field of HNVf assessment and monitoring. In particular, by providing an approach to analyze the location and changes over time of HNVf types in relation to areas under distinct legal protection (such as the Natura 2000 network), it can help assess the role that such nature conservation designations have in protecting HNVf and indicate where additional agricultural or nature conservation policy and support mechanisms may need to be targeted.