The worldwide increase of citrus cultivation has contributed a lot in the emergence of various diseases. Gummosis is one of the most important disease of citrus caused by several species of Botryosphaeriacae most prominent Lasiodiplodia spp. along with some other species of genus Phytophthora and Fusarium. The objective of this study was the molecular identification and characterization of pathogens associated with citrus gummosis from major citrus growing areas of Punjab, Pakistan. This study included collection of diseased samples followed by isolation, morphological identification, molecular characterization, pathogenicity and virulence comparison of associated fungal pathogens. To identify the causal pathogens of citrus gummosis, symptomatic diseased bark tissues were collected from thirty localities of five major citrus production areas of Punjab. Lasiodiplodia spp. (Lasiodiplodia pseudotheobromae, Lasiodiplodia theobromae) Fusarium spp. (Fusarium equiseti, Fusarium solani, Fusarium lateritium, Fusarium proliferatum) were isolated from diseased samples. Preliminary identification of isolated fungi was based on morphological and cultural characters. For molecular identification and characterization nucleotide sequences of the ITS region (ITS1-5.8S-ITS2) and partial sequence of the β-tubulin gene was targeted. Pathogenicity analysis of all the isolated species were performed on Citrus jambhiri cultivar which is commonly used rootstock in Punjab. L. pseudotheobromae and L. theobromae both produced typical symptoms of citrus gummosis. L. pseudotheobromae found more virulent than L. theobromae. These findings confirm that L. pseudotheobromae and L. theobromae are primary cause of citrus gummosis in Punjab, Pakistan. Fusarium species failed to produce any symptoms. Fusarium spp. has no role in disease development however it could be a secondary invader or saprophytic. Although association of different species of Phytophthora genus with this disease is reported from different parts of the world but it was not successfully isolated after numerous recommended attempts. This study provides new information for future studies of disease management, quarantine programs and especially the development of resistant varieties against citrus gummosis.