The maize CMS-S near isogenic line (NIL) developed by author and the backcross progeny (BC1) derived from it were used to identify molecular markers linked to the Rf3 gene and subsequently determine its chromosomal location on the linkage map of maize. Bulk segreant analysis was performed using AFLP technique. From the survey of AFLP primer combination, two AFLP markers, (EcoRI-AGG/MseI-CAC and EcoRI-AAC/MseI-CAG), which were named RR6 and RR7 respectively, linked to the Rf3 gene were identified. However, AFLP marker RR6 showed polymorphism between parents, and bulks were used to survey the available 100 individuals of the BC1 population, 2 out of 100 shed recombination. The recombination-rate was 2%. The genetic distance between Rf3 gene and AFLP marker RR6, was approximately 2.0 cM. And then, the RR6 was successfully cloned and sequenced, primer synthesized and converted to SCAR marker so that PCR marker can be developed for the marker-assisted selection. In RFLP analysis, marker RR6 linked to Rf3 was found to be located between RFLP loci asg20 and php20581b, and mapped on chromosome 2L.