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Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. In recent decades, OS treatment has reached a plateau and drug resistance is still a major challenge. Therefore, the present study aimed to analyze the expression of the genes related to pharmacogenetics in OS. The expression of 32 target genes in 80 paired specimens (pre-chemotherapeutic primary tumor, post-chemotherapeutic primary tumor and pulmonary metastasis) obtained from 33 patients diagnosed with OS were analyzed by the real-time PCR methodology. As the calibrators (control), five normal bone specimens were used. The present study identified associations between the OS outcome and the expression of the genes <i>TOP2A</i>, <i>DHFR</i>, <i>MTHFR</i>, <i>BCL2L1</i>, <i>CASP3</i>, <i>FASLG</i>, <i>GSTM3</i>, <i>SOD1</i>, ABCC1, <i>ABCC2</i>, <i>ABCC3</i>, <i>ABCC5</i>, <i>ABCC6</i>, <i>ABCC10</i>, <i>ABCC11</i>, <i>ABCG2</i>, <i>RALBP1</i>, <i>SLC19A1</i>, <i>SLC22A1</i>, <i>ERCC1</i> and <i>MSH2</i>. In addition, the expression of the <i>ABCC10</i>, <i>GGH</i>, <i>GSTM3</i> and <i>SLC22A1</i> genes were associated with the disease event, and the metastasis specimens showed a high expression profile of <i>ABCC1</i>, <i>ABCC3</i> and <i>ABCC4</i> genes and a low expression of <i>SLC22A1</i> and <i>ABCC10</i> genes, which is possibly an important factor for resistance in OS metastasis. Therefore, our findings may, in the future, contribute to clinical management as prognostic factors as well as possible therapeutic targets.