Tomato leaf spot, caused by the ascomycete fungi Stemphylium spp., including S. lycopersici, S. solani and S. vesicarium, is a severe threat to tomato production worldwide. In Uruguay, the disease has become more prevalent in both field and greenhouse production systems in recent years, primarily due to the use of susceptible tomato varieties. This study focuses on the identification, morphological characteristics, genetic diversity and virulence of Stemphylium isolates responsible for leaf spot in Uruguay's major tomato-producing regions. Phylogenetic analysis using partial sequences of the rDNA internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) genes showed that S. lycopersici is the predominant pathogen, with 33 isolates identified, whereas only three isolates were classified as S. vesicarium. Genetic diversity analysis using inter simple sequence repeat (ISSR) markers identified two main clusters of S. lycopersici isolates that correlate with distinct geographical regions. Cultural and morphological characteristics of S. lycopersici varied significantly and pathogenicity assays revealed a broad spectrum of virulence among S. lycopersici isolates, with some isolates causing extensive necrosis and others showing minimal disease lesions. S. lycopersici primarily penetrates plant tissues through stomata and trichomes, and hyphae proliferate both within and on plant tissues, causing chlorosis and necrosis. Infected tissues exhibit alterations in cell wall composition associated with the incorporation of phenolic compounds. This is the first report characterizing various isolates of S. lycopersici and S. vesicarium responsible for tomato leaf spot in Uruguay.