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Abstract Methotrexate is a widely used drug in clinical practice for the treatment of collagen vascular diseases and malignant tumors. It has good anti-inflammatory and anti-proliferative effects, but the cytotoxicity of methotrexate can cause various adverse reactions in patients. Studies have shown that the sensitivity and tolerance of different individuals to methotrexate is different. There are many reasons for this difference. Among them, genetic polymorphism is one of the main factors that cause individual differences. This article provides an overview of the genetic polymorphisms of key proteins involved in methotrexate metabolism and transport, such as MTHFR, FPGS, γ-GGH, ABC transporter, OATPs, SLC, TS and DHFR, are related to their efficacy and adverse reactions. The aim is to clarify the impact of genetic polymorphisms on the efficacy and adverse effects of methotrexate at the pharmacogenomic level, in order to provide a basis for the clinical application of methotrexate.