We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.