Osteosarcoma is characterized by diverse genetic mutations, including single-nucleotide variants (SNVs), which can complicate clinical outcomes of the treatment. This study identified key mutations or polymorphisms in genes that correlate with osteosarcoma prognoses. A total of 110 patients with osteosarcoma were assigned to “good” or “poor” cohorts depending on their 5-year disease-free survival (DFS) after surgery and chemotherapeutic treatment. We performed next-generation sequencing analysis of tumor tissues for prognosis-associated SNVs in 315 tumorigenesis-related genes, followed by modeling of clinical outcomes for these patients using random forest classification via a support vector machine (SVM). Data from the Chinese Millionome Database were used to compare SNV frequency in osteosarcoma patients and healthy people. SVM screening identified 17 nonsynonymous SNVs located in 15 genes, of which rs17224367 and rs3733406 (located in MSH2 and FAT1, respectively) were strongly correlated with osteosarcoma prognosis. These results were verified in a 26-patient validation cohort, confirming that these SNVs could be used to predict prognosis. These results demonstrated that two SNVs located in MSH2 and FAT1 are associated with prognosis of osteosarcoma patients.