For eukaryotic pathogens that have low levels of genetic variation, multilocus microsatellite typing (MLMT) offers an accurate and reproducible method of characterizing genetic diversity. Here, we describe the application of an MLMT system to the emerging pathogenic fungus Penicillium marneffei. Isolates used for this study were those held in the culture collections of the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and the Chiang Mai University Department of Microbiology, Chang Mai, Thailand. High genetic diversity and extensive spatial structure were observed among clinical isolates, with the geographical area of origin for each isolate strongly correlating with the occurrence of two deeply divided clades. Within each clade, multilocus linkage associations were highly significant and could be explained by genetically differentiated populations or by an exclusively clonal reproductive mode, or both. Our results show that southeast Asian penicilliosis is caused by a fungus with a complex population genetic structure. Furthermore, this MLMT system generates digital data that can be easily queried against a centrally held database via the internet (http://pmarneffei.multilocus.net/); this provides a powerful epidemiological tool for analyzing the underlying parameters that are responsible for the emergence of P. marneffei in human immunodeficiency virus-positive populations.