To characterize the fungal pathogen responsible for loquat scab and establish differences in pathogenicity in loquat, eight strains identified as Fusicladium eriobotryae were isolated from either loquat leaves or fruit showing scab symptoms in Spain. Loquat plants belonging to the cv. Peluche were infected via a newly developed infection system that was based on spraying susceptible loquat plants with fungal spore suspensions, keeping the plants for 1 week in 100% humidity, and then transferring the plants to ambient relative humidity in greenhouses. Scab symptoms were analyzed and pathogenic characterization of all F. eriobotryae strains revealed different degrees of aggressiveness. Based on infection progression and severity of scab symptoms, strain ST1 was confirmed as the most aggressive in cultivars in the Mediterranean region. Strain ST1, which even caused chlorotic spots in loquat stems, is so aggressive it can be used to identify highly resistant cultivars using this in vivo system. Molecular characterization of internal transcribed spacer ribosomal DNA and, particularly, the glyceraldehyde 3-phosphate dehydrogenase gene, clearly distinguished loquat strains from Venturia inaequalis. Moreover, random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction techniques were used to qualitatively discriminate between species and report the variations within fungal populations. Molecular variability was checked by comparing all the different strains and enabled the specific identification of F. eriobotryae. Although no association was observed between any pattern and phenotypic traits, such as aggressiveness, RAPD provided a specific profile that allowed fungal identification.