Abstract: Objective: The purpose of this paper is to clone leukemia associated gene and investigates aberrant DNA methylation of this gene in acute myeloid leukemia (AML). Methods: The Restriction landmark genomic scanning (RLGS) and Southern Blot were performed on genomic DNA from AML bone marrow (BM) samples. Results: An abnormal 221 bp fragment due to a distinct DNA methylation on NotI site was cloned. Sequence analysis showed that the fragment represented a portion of exon 2 of the WIT-1 gene. Seven of 21 (33.3%) chemosensitive AML and 7 of 8 (87.5%) primary refractory AML showed methylation of WIT-1 gene. The incidence of WIT-1 methylation in primary refractory AML was significantly higher than that noted in chemosensitive AML.Conclusion: The results indicate that RLGS is a effective method to find leukemia associated gene in AML and that WIT-1 gene methylation analysis is important to estimate therapeutic response in AML.