Abstract:To identify walnut-associated Alternaria spp., the cultural characteristics, sporulation phenotypes and conidial characteristics were described from cultures grown on potato carrot agar (PCA). Meanwhile, the phylogenetic analysis was carried out based on the sequences of ITS, OPA2-1 and OPA1-3 anonymous genomic regions. The results released that 33 isolates of total 34 tested isolates were A.alternata and only one isolate ‘10-19’ was A.tenuissima. Sporulation pattern of A.alternata isolates appeared as low bushy clumps of well-branched conidial chains (4 to 10 spores); conidia varied from obpyriform to ovate, the average size of spore body 25.6 μm×7.4 μm, with 2-8 transverse. A.tenuissima isolate produced long conidial chains (about 10 spores) with occasional branching forming secondary chains (1-5 spores); Conidia were obclavate or ellipsoidal with 1-6 transverse septa, central 1-2 septa often thicker and darker than the others, with the average size of spore body 26.3 μm×13.5 μm. The phylogenetic analysis was showed the sequences of ITS, OPA2-1 and OPA1-3 anonymous genomic regions can be used to distinguish the small-spores Alternaria species to some extent. A.alternata was dominant species of walnut-associated Alternaria spp.