The molecular karyotype of a series of Giardia lamblia isolates representing the two major genotypes (Groups 1 and 3) was generated by assigning 13 genetic markers to chromosomes separated by pulsed-field gel electrophoresis. The co-localization identified five linked groups of genetic markers in Group 1 isolates. For each of the five linkage groups, there were up to four size variants that hybridized with the same genetic markers. Long range physical maps of the regions flanking the low copy number genetic markers indicated that these size variants were homologous chromosomes. The linkage groups were similar in Group 1 and 3 isolates. The core of each chromosome was stable while the subtelomeres were variable. The location of the ribosomal DNA repeats was variable among the different isolates and they were found in the subtelomeric regions of any of the five linkage groups. The data suggest a functional ploidy of at least four. Hypervariable subtelomeric regions of homologous chromosomes provide the structural basis of the chromosome size heterogeneity that is characteristic of G. lamblia.