Abstract: We have identified four new genetic loci: CHL2 (on chromosome XII), CHL3 (on chromosome XII); CHL4 (on chromosome IV), and CHL5 (on chromosome IX), controlling mitotic transmission of yeast chromosomes. The frequency of loss of chromosomes is 10-100-fold higher in chl5, chl2, chl3 and chl4 mutants than observed in wild-type strains. The mutants also show unstable maintenance of artificial circular minichromosomes with various chromosomal replicators (ARS) and one of the centromeric loci (CEN3, CEN4, CEN5 or CEN6). The instability of minichromosomes in the chl5, chl2, and chl4 mutants is due to the loss of minichromosomes in mitosis (1:0 segregation). In the chl3 mutant the instability of artificial minichromosomes is due to nondisjunction (2:0 segregation). The CHL3 gene therefore appears to affect the segregation of chromosomes during cell division.