Benign fibro-osseous lesions of bone (BFOL) comprise a group of clinically distinct entities with significant histologic overlap and often occur in children and adolescents. Because of prior studies indicating that these lesions possess distinct karyotypic abnormalities, we conducted a retrospective review of cytogenetic analyses performed in a series of 16 BFOL in children and adolescents diagnosed at two institutions. These comprised five cases with the diagnosis of ossifying fibroma, four with osteofibrous dysplasia, and seven with fibrous dysplasia arising in the skeleton of 16 children and adolescents. All cases were analyzed using standard G-banding techniques on fresh tumors explanted in tissue culture media. Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) were used to analyze selected metaphases of a talar lesion with the histologic features of ossifying fibroma. All four confirmed ossifying fibromas, including the talar lesion, contained clonal aberrations fusing breakpoints on Xq26 and 2q33, and one case with dissimilar histology did not. Three of four osteofibrous dysplasias contained multiple copies of chromosomes 8, 12, and/or 21. All but two fibrous dysplasia cases exhibited either a completely normal karyotype or single cell aberrations. One fibrous dysplasia had subtle chromosomal abnormalities not seen in other cases in the series, and another had complex abnormalities involving multiple chromosomes. Our current and published results indicate that cytogenetics might be of ancillary use in the diagnosis of BFOL and that a characteristic chromosomal arrangement is associated with ossifying fibroma.