<p style="text-align: justify;"><strong>Aim</strong>: The control of geographical origin is one of the most challenging topics regarding wine authenticity. The aim of the present study was to assess the <sup>87</sup>Sr/<sup>86</sup>Sr ratio of vineyard soils from Portuguese Denominations of Origin (DO) and evaluate its suitability as a tool for origin authentication.</p><p style="text-align: justify;"><strong>Methods and results</strong>: An analytical protocol was optimized (chromatographic separation of Sr and Rb, followed by inductively coupled plasma-mass spectrometry (ICP-MS) analysis) for <sup>87</sup>Sr/<sup>86</sup>Sr isotopic ratio determination in soil-wine system. The <sup>87</sup>Sr/<sup>86</sup>Sr ratios of soils from four vineyards located in three Portuguese DO (Dão, Óbidos and Palmela), established on distinct soil types, were determined. Significant differences were found between soils of different DO regions. The soil in the Dão DO, developed on granites, showed a statistically higher <sup>87</sup>Sr/<sup>86</sup>Sr ratio than the other soils, which were developed on sedimentary formations.</p><p style="text-align: justify;"><strong>Conclusion</strong>: The results show clearly that <sup>87</sup>Sr/<sup>86</sup>Sr ratio may represent a suitable fingerprint for these Portuguese DO.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This study highlights the relevance of setting up an international databank of <sup>87</sup>Sr/<sup>86</sup>Sr values for use for geographical identification and authentication.</p>