Alternaria alternata is one of the most important fungi causing brown spot on tangerine. Samples of A. alternata were collected from different cultivars of tangerine affected by brown spot in orchards in Jahrom region, Iran. Samples were cultured on potato dextrose agar (PDA) media and purified using the single-spore method. Pathogenicity tests were done based on Koch's postulates on healthy and scratched leaves of different tangerine cultivars. All isolates were pathogenic on both healthy and scratched leaves. Ponkan and Mahalli (local isolates) showed the greatest growth, while Fortune and Kinnow isolates had the least growth on PDA media. The rDNA ITS1 region of Clementine, Mahalli, Ponkan, Lee, Fortune, Kinnow, Page, and Osceola isolates was sequenced. One PCR product (360 bp) was recovered from all mycelia multiplications. Sequences of all isolates were deposited in GenBank. BLAST searches of the NCBI database showed that the closest matches were A. alternata. The morphological tests were supported by molecular experiments. The phylogenetic tree of the sequences was mapped based on neighbour-joining procedures. Grouping of all isolate sequences placed them in two main clusters. Mahalli isolates in cluster 2 were in a different group from the other isolates. In general, the results showed intra-species genetic differences in rDNA ITS1 sequences among A. alternata isolates collected from tangerine cultivars. In this study, A. alternata was identified as the cause of Alternaria brown spot of tangerine cultivars in southern Iran.