Sweet chestnut (Castanea sativa Mill.) is a nutritious food with high social and economic impact in Portugal. Fungal infection and rots lead to great economic and quality losses in stored chestnut. The fungus Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) is currently considered one of the major threats to the chestnut chain worldwide. Considering the lack of knowledge on both the disease and the causal agent in Portugal, studies have been conducted in an attempt to timely develop the necessary control strategies towards the mitigation of the disease. Under this scope, the chestnut brown rot and its causal agent have been characterized for the first time in Portugal. The study was performed on chestnuts from Braganca, Portugal, that were received, processed and stored in a local industry during the growing season 2018-2019. Thirty-three samples were collected from different processing stages and from three chestnut varieties (Longal, Judia and Martaínha). Several isolates of G. smithogilvyi obtained were characterized at the morphological, ecophysiological, enzymatic and molecular levels. The fungus was also characterized in terms of pathogenicity and virulence. G. smithogilvyi was confirmed as the causal agent of brown rot disease in Portuguese chestnut varieties, which showed high susceptibility. The fungus showed high adaptability to chestnut substrates. The Portuguese isolates of G. smithogilvyi are morphologically and genetically similar to those isolated in other countries, even though some physiological and enzymatic variability was observed among the Portuguese isolates. Post-harvest control strategies towards the mitigation of the disease are discussed.