In order to identify candidate tumor suppressor genes (TSGs) in childhood acute lymphoblastic leukemia (ALL), firstly, loss of heterozygosity (LOH) of 6q16.3～21 in 139 primary ALL samples was analyzed by using polymerase chain reaction (PCR) and 11 microsatellite markers. The frequency of LOH on 6q16.3～21 was 32%. A 2-cM high frequency deletion region was flanked by D6S1709 and D6S301 loci at 6q16.3～21. Clinical data showed that patient with 6q16.3～21 LOH had higher WBC counts and blast cells (P < 0.05). The statistics about age, sex, classification of morphology and immunology were indistinct (P > 0.05). Then, positional cloning strategy, bioinformatics technology and reverse transcription-polymerase chain reaction (RT-PCR) were used to identify candidate TSGs and its cDNA fragments at 6q16.3～21, especially at the high frequency deletion region. Comparing with expression of normal peripheral blood mononuclear cell, EST screened in D6S1709-D6S301(GenBank Accession No.AA403058) was down-regulation in ten of fifteen childhood ALL (P < 0.05). Digital differential display showed that the expression levels of AMD1, PPIL6 and WASF1 were lower in cancer tissues than in normal tissues(P < 0.05). These findings may provide new clues in cloning of childhood ALL TSGs at 6q16.3～21.