Chromosome painting using commercially available human chromosome-specific DNA libraries was performed to elucidate chromosomal rearrangements in lemur evolution. Human-specific probes for chromosomes 3, 14, 15, and 21 were used to paint chromosomes of six species: Eulemur fulvus mayottensis, Varecia variegata, Lemur catta, Hapalemur simus, H. griseus griseus, and H. aureus. All human chromosome libraries hybridized specifically to chromosome segments of varying length or to whole arms of Lemur chromosomes. The labeling was clearly visualized and permitted precise delineation of the hybridized Lemur chromosomal segments. The use of commercial probes of human chromosomes for chromosome painting appears efficient enough to investigate homology in different species of Lemur. In general, the results obtained by chromosome painting in this study confirm results previously obtained by the R-banding technique but modify the location of some chromosomal rearrangements on different branches of the evolutionary tree of the Lemuridae and reveal some new rearrangements that were not detectable with banding techniques. These results show that chromosome painting with human chromosome-specific DNA libraries can provide useful information in comparative studies on karyotypes of distantly related mammalian species, providing a powerful tool for evolutionary studies, especially in phylogeny.