Abstract:A biotin-labeled in situ hybridization technique was used in order to physically map two RFLP markers-umc22 and umc122-tightly linked to the Htl gene on the chromosomes of maize ( Zea mays L. ). The results showed that both markers located on the chromosomes 2,7 and umc22 also hybridized with chromosome 4, which demonstrated that the two markers were a duplicated or triplicated sequence. The average detection rate of in situ hybridization was 17.46%. The percent distances of umc22 and umc122 from centromere on the chromosome 2 were 58.36 ± 3.19 and 61.02 ± 4.32 respectively, and on the chromosome 7 were 44.70 ± 2.11 and 45.19 ± 2.27 respectively, which indicates that there are no differences between genetic and physical distances of two markers umc22 and umc122. It was deduced that the gene Htl should also have its homeologous sequence between the hybridization sites of umc22 and umc122 on 7 L besides its location between the two hybridization sites.