ABSTRACT A collection 160 isolates of Cercospora kikuchii was made from South America and 245 from Japan. DNA fingerprint patterns were analyzed based on amplified fragment length polymorphism among the sample isolates, dividing the isolates into seven lineages (I to VII). Partial nucleotide sequence analyses of the beta-tubulin gene supported this division into seven lineages. Lineages I and III commonly existed in South America and Japan. In all, 136 of the 160 isolates from South America and 223 of the 245 isolates from Japan belonged to lineage I, indicating that lineage I was the major lineage in each area; 5 isolates from South America and 8 isolates from Japan belonged to lineage III. Lineages II (12 isolates) and IV (2 isolates) were specific to Japan and lineages V (3 isolates), VI (1 isolate), and VII (15 isolates) specifically existed in South America. These results suggest that the population genetic structure of C. kikuchii was different between South America and Japan, but the dominance of lineage I was common between the two areas.