Osteogenic sarcoma (OS) is the most common malignant bone tumor in children and adolescents. Despite advances in molecular genetic characterization of pediatric and adult tumors, the diagnosis of OS still depends almost entirely on light microscopy. The lack of consistent genetic changes in OS has greatly hindered the development of any diagnostic molecular test. Recently, whole-genome sequencing has shown that ~50% of cases of OS have a translocation involving the TP53 gene with breakpoints confined to the first intron. We developed a 2 color break-apart fluorescent in situ hybridization (FISH) probe for intron 1 of TP53 and applied it to an archived series to assess its diagnostic utility. The study group included 37 cases of OS (including osteoblastic, chondroblastic, and fibroblastic), as well as 53 cases of non-OS pediatric sarcomas (including Ewing sarcoma, rhabdomyosarcoma, undifferentiated small cell sarcoma, CCNB3-BCOR sarcoma, CIC-DUX sarcoma, synovial sarcoma, and malignant peripheral nerve sheath tumor) and 27 cases of benign bone lesions (including osteoblastoma, chondromyxoid fibroma, fibrous dysplasia, and fibro-osseous dysplasia). A rearranged signal was found in 20/37 cases (54%) of OS and in none of the other sarcomas or benign bone lesions, giving the FISH test 100% specificity for a diagnosis of OS. p53 immunostaining was generally not predictive of the results obtained by FISH and could not substitute for this test. This FISH probe offers a simple and specific genetic test to aid in the diagnosis of OS, despite the genetic complexity of this tumor.