Mango is one of the most popular and nutritious fruits in the world and Mexico is the world’s largest exporter. There are many diseases that directly affect fruit yield and quality. During the period 2016–2017, leaves with grey leaf spots were collected from 28 commercial mango orchards distributed in two main production areas in Sinaloa State of Mexico, and 50 Neopestalotiopsis isolates were obtained. Fungal identification of 20 representative isolates was performed using morphological characterization and phylogenetic analysis based on the internal transcribed spacer (ITS) region of ribosomal DNA, part of the translation elongation factor 1-alpha (TEF) and the β-tubulin (TUB) genes. Phylogenetic analysis indicated that the 20 isolates from this study formed four consistent groups, however, overall tree topologies do not consistently provide a stable and sufficient resolution. Therefore, even though morphological and phylogenetic separation is evident, these isolates were not assigned to any new taxa and were tentatively placed into four clades (clades A–D). Pathogenicity tests on detached mango leaves of cv. Kent showed that the 20 isolates that belong to the four Neopestalotiopsis clades from this study and induce lesions on mango leaves. This is the first report of species of Neopestalotiopsis causing mango grey leaf spot disease in Mexico.