Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Despite its importance, our knowledge of the epidemiology of cryptococcosis in Japan remains limited. To establish an epidemiological database on cryptococcosis in Japan, we determined the genetic variability of 44 Japanese clinical isolates of C. neoformans (var. grubii: serotype A) by multilocus sequence typing (MLST). The strains were clinically isolated from 1992 to 2011 in 5 different areas of Japan (the Hokkaido region [n = 1], Kanto region [n = 32], Chubu region [n = 1], Kansai region [n = 1], and Kyushu region [n = 9]). According to the method recommended by the International Society for Human and Animal Mycology cryptococcal genotyping working group, 36 isolates (82%) were identified as sequence type (ST)46. The remaining strains belonged to ST45 (n = 1) and ST47 (n = 1), and 6 isolates belonged to novel independent STs. There was little geographic difference in the ST population. Our present data are still limited; however, because most clinical isolates showed the same MLST profile in Japan, applying the current MLST scheme for Cryptococcus may at times be insufficient for investigating the infection route among outbreak cases. To solve this problem, it may be necessary to investigate other gene loci or develop a novel method with greater discriminatory power. However, in cases in which a strain belongs to a minor ST, our data may serve as useful epidemiological information in Japan.