Several lines of evidence suggest that defects in telomere maintenance play a significant role in the initiation of genomic instability during carcinogenesis. Although the general concept of defective telomere maintenance initiating genomic instability has been acknowledged, there remains a critical gap in the direct evidence of telomere dysfunction in human solid tumors. To address this topic, we devised a multiplex PCR-based assay, termed TAR (telomere-associated repeat) fusion PCR, to detect and analyze chromosome end-to-end associations (telomere fusions) within human breast tumor tissue. Using TAR fusion PCR, we found that human breast lesions, but not normal breast tissues from healthy volunteers, contained telomere fusions. Telomere fusions were detected at similar frequencies during early ductal carcinoma in situ and in the later invasive ductal carcinoma stage. Our results provide direct evidence that telomere fusions are present in human breast tumor tissue and suggest that telomere dysfunction may be an important component of the genomic instability observed in this cancer. Development of this robust method that allows identification of these genetic aberrations (telomere fusions) is anticipated to be a valuable tool for dissecting mechanisms of telomere dysfunction.