Asparagine synthetase (ASNS) is an enzyme expressed ubiquitously in mammalian cells. Here, we discovered two 14-bp tandem repeat (2R, wild-type) sequences in the first intron of the gene. The 14-bp sequence is similar to the three GC-boxes (GC-I, -II, and -III) found in the promoter region of the ASNS gene, as well as, the binding site of transcription factor Sp-1. Approximately 75% of acute lymphoblastic leukemia (ALL) samples had the 2R sequence in both allele; however, 20% and 3% ALL samples had three (3R) and four (4R) 14-bp tandem repeats in one allele, respectively; the other allele had 2R. The tandem repeat sequence was not specific to the leukemia cells but represents a novel germline polymorphism. Interestingly, the 14-bp sequence functioned as a transcriptional enhancer element as shown by reporter analysis and formed a protein-DNA complex in vitro. Our data for the first time show that the ASNS gene has tandem repeated sequences as a polymorphism, and it can function as a transcriptional element; increased number of tandem repeat producing increased activity. Clinical significance in ALL requires further studies.