Recent molecular investigations have demonstrated over-expression of a large number of tumor associated antigens (TAAs) in a variety of malignancies. Over-expression of ROR1 gene, a member of the receptor tyrosine kinase family, has recently been reported in B-cell chronic lymphocytic leukemia. Wilms' tumor gene 1 (WT1) has long been known as a universal TAA expressed in a variety of solid and hematopoietic malignancies. In the present study, the expression profile of ROR1 and WT1 was investigated in different immunophenotypic subsets of B-cell acute lymphoblastic leukemia (B-ALL) patients. RT-PCR method was used to determine the ROR1 and WT1 genes expression in bone marrow (BM) and peripheral blood (PB) samples from 51 newly diagnosed Iranian B-ALL patients. Isolated tumor cells from all patients were immunophenotyped by flow cytometry. Based on immunophenotypic results, our B-ALL patients were classified in four differentiation subsets; Pro-B (n = 7), Pre-B I (n = 29), Pre-B II (n = 13) and Immature/mature B-ALL (n = 2). Although ROR1 was over-expressed in more mature subsets (16.7%, 42.9%, 45.5% and 100%, respectively), WT1 was more represented in immature subsets of B-ALL patients (57.1%, 64.3%, 38.5% and 0%, respectively). Comparison of the frequency of ROR1 and WT1 positive samples at each immunophenotypic subtype revealed statistically significant difference only in Pre B I subtype (p = 0.02). Our results suggest that expression of ROR1 and WT1 in B-ALL is associated with the differentiation stage of the leukemic cells.