To address whether mutations and single nucleotide polymorphisms (SNPs) in perforin gene (PRF1) are correlated with acute lymphoblastic leukemia (ALL) in Chinese, we screened mutations in codon region of PRF1 in 111 ALL patients, and correlated the results with patients' immunophenotype, karyotype and fusion genes. Four novel monoallelic missense and two novel monoallelic synonymous mutations (G198R, R225Q, D486G, R509K, S388S and Q540Q) were identified in 9 B-ALL, of whom 7 cases carried BCR-ABL gene, one carried MLL-AF4 fusion gene, and one lost two chromosomes. Our results suggest that mutations in PRF1 may play a role in the pathogenesis of B-ALL.