Aims: Methotrexate (MTX) is a basic therapy for juvenile idiopathic arthritis (JIA). MTX intolerance can significantly impact quality of life, treatment adherence and outcomes. We aimed to assess the prevalence of MTX intolerance and to identify clinical factors associated with intolerance in children and adolescents with JIA. Methods: This cross-sectional study was conducted at a large pediatric rheumatology referral center between July 2019 and July 2021. It included 94 patients with JIA, aged up to 19 years, who had been treated with MTX (oral or subcutaneous) for at least three months. Patients with systemic JIA and those exhibiting toxic MTX effects were excluded. Demographic and clinical data were collected, and MTX intolerance was assessed using the Methotrexate Intolerance Severity Score (MISS) questionnaire. MTX intolerance was defined as a total MISS score of ≥6, including at least one anticipatory, associative, or behavioral symptom. Statistical analyses were performed to compare MTX-tolerant and MTX-intolerant groups. Results: The median patient age was 9.9 years (range 2.3–18.5 years), and 69 (73.4%) were female. The median age at disease onset was 3.7 years, and the median duration of MTX therapy was 2.3 years. The prevalence of MTX intolerance was 24.5%. The most common symptom was nausea after MTX intake (38.3%), followed by behavioral problems such as irritability or restlessness. Intolerant patients were significantly older at disease onset and MTX initiation (U=577.0, p=0.04 and U=555.5, p=0.02, respectively), and MTX as first-line therapy was more frequent in this group (X2=5.78, p=0.02). There was a strong positive correlation between age at disease onset and MTX initiation (r=0.9, p<0.001). Conclusions: MTX intolerance is relatively common in pediatric patients with JIA and is associated with older age at disease onset and MTX initiation, as well as the use of MTX as first-line therapy.