© 2018 2018 Future Medicine Ltd. Aim: A third of rheumatoid arthritis patients discontinue methotrexate treatment due to inefficacy or toxic side effects. Recently, an association between SLC04A1 rs2236553, SLC22A2 rs624249 and rs316019, and SLC28A2 rs10519020 and rs1060896 with the efficacy and toxicity of methotrexate was reported. This study aims to replicate these findings in an independent cohort (n = 324). Methods: Regression analyses tested the associations between genotype and methotrexate response or toxicity. Results: In the discovery study, there was a significant association between toxicity and rs624249, and rs1060896. These associations were not replicated in the independent cohort. Neither study observed an association between methotrexate efficacy and SLC04A1, SLC22A2 or SLC28A2 variants. Conclusion: Current evidence does not support associations between variants in SLC04A1, SLC22A2 and SLC28A2 with methotrexate efficacy or toxicity.