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A decline of various grapevines (<i>Vitis vinifera</i> L.) in the province of Doukkala in Morocco was observed in 2021. The causal pathogen was identified as <i>Lasiodiplodia viticola</i> based on morphological characteristics and phylogenetic analysis of the internal transcribed region (ITS), the β-tubulin gene (TUB) and calmodulin (cmdA). Koch’s postulates were confirmed by successful re-isolation of <i>L. viticola</i> from plants inoculated with the pathogen under controlled conditions. The disease was shown to be prevalent in Bni Hilal (71.43%), Laamria (60%), and Boulaouane (40%) districts, but was quasi-absent in Lmechrek. To understand the dominance of <i>L. viticola</i> as one of the grapevine trunk pathogens, effects of temperature (10–40 °C) and pH (pH 3–pH 12) on growth and sporulation were investigated. The species were able to grow in a range of temperatures ranging from 15 to 40°C and showed a higher growth rate at 35 °C. The fungus were also characterized by a broad optimum pH ranging between 3–12. This study is the first report dealing with <i>L. viticola</i> associated with grapevine trunk diseases in Morocco. Additional studies are therefore required to understand the high occurrence of this disease in vineyards, which is likely due to climate changes. A good understanding of this complex disease might help to develop a reliable and sustainable preventive control strategy.