Osteosarcoma (OS) is the most common malignant bone tumor affecting adolescents and young adults and it usually occurs in the long bones of the extremities. The detection of cancer-related genetic alterations has a growing effect in guiding diagnosis, prognosis and targeted therapies. However, little is known about the molecular aspects involved in the etiology and progression of OS, which limits options for targeted therapies. The present study described a case of an adolescent patient (16-years-old) who was diagnosed with conventional central OS in the right distal femur without the evidence of pulmonary metastases; the patient was treated with surgery and adjuvant chemotherapy. Genetic alterations in resected tumor tissue were investigated via next-generation sequencing (NGS) technology using a targeted NGS panel. Sanger sequencing was also performed to investigate somatic and germline TP53 mutations (exons 4-8). NGS analysis revealed an intratumor heterogeneity signature in OS tumor, including several single nucleotide variants identified in genes encoding tyrosine kinase proteins. No PCR products for TP53 exon 5 were detected in the tumor sample by PCR analysis prior to Sanger sequencing, suggesting a significant deletion in this exon. Sanger sequencing analysis revealed the missense variant TP53 c.712T>A (p.Cys238Ser) in tumor tissue sample, thus reinforcing the role of TP53 somatic mutations in OS development. Additionally, the TP53 c.215C>G (p.Pro72Arg) germline missense variant was identified in the peripheral blood sample. In conclusion, the findings provided new information on genetic aspects that may contribute to OS development, especially in pediatric patients.