The rat osteosarcoma cell line UMR-106 is widely used for the study of bone cancer biology. We have characterized UMR-106 with a combination of optical genome mapping (OGM), long-read sequencing (for identification of variants and 5mC methylation), and short-read RNA sequencing for expression. Genome sequence was compared to a Sprague-Dawley control animal, the strain from which UMR-106 was derived, and expression data were compared to a public rat osteoblast dataset. Using the COSMIC database to identify the most affected genes in human osteosarcomas (OS) we found somatic mutations in Tp53 and H3f3a in UMR-106. OGM identified a relatively small number of differences between the cell line and a strain-matched control animal but did detect a ~45 Mb block of amplification that included Myc on chromosome 7 which was confirmed by long-read sequencing. The amplified region showed several blocks of non-contiguous rearranged sequence implying complex rearrangements during their formation and included 14 genes reported as biomarkers in human osteosarcoma, many of which also showed increased transcription. A comparison of 5mC methylation from the nanopore reads of tumor and control identified several genes with distinct differences including the OS marker Cdkn2a. This dataset illustrates the value of long DNA methods for the characterization of cell lines and how inter-species analysis can inform about the genetic nature underlying mutations that underpin specific tumor types. The data should be a valuable resource for investigators studying osteosarcoma, in general, and specifically the UMR-106 model.