Cartilaginous neoplasms are often histologically and therapeutically challenging. Predicting biologic behavior can be difficult. In this study, 120 nonneoplastic, benign, and malignant cartilaginous lesions from 103 patients were cytogenetically analyzed in a 6-year period after short-term culture. For selected cases, fluorescent in situ hybridization (FISH) techniques using chromosome-specific probes were performed on metaphase/interphase preparations and on paraffin-embedded tissue sections. Clonal abnormalities of chromosomes 2, 3, 5, 7, 8, and 12 were most frequently observed. Involvement of chromosomes 5, 8, and 12 may be etiologically significant because of the gene localizations for the human cartilage link protein, Langer-Giedion syndrome (a rare syndrome characterized by multiple exostoses), and type II collagen (a major component of normal cartilage) respectively, to these three chromosomes. That chromosome 7 abnormalities were observed only in malignant tumors is of diagnostic value. The identity of three marker chromosomes and the significance of trisomy 7 (a finding of controversial meaning), were determined with FISH. That the presence of chromosome aberrations and increasing histologic grade strongly correlated (p = 0.001) is of prognostic importance. Moreover, complex aberrations were observed nearly exclusively in high-grade tumors (p = 0.001). The data show that nonrandom chromosome loci are aberrantly affected in cartilaginous lesions and that these abnormalities may be of significant histopathogenetic consequence. In addition, these chromosome abnormalities appear to be diagnostically and prognostically valuable in classifying and grading chondromatous neoplasms.