Background: This study aimed to the serological and molecular diagnosis of Toxoplasma gondii infections and related risk factors in patients with thalassemia major and healthy controls. Methods: This case-control study was performed in Shahrekord University of Medical Sciences, Shahrekord, west of Iran from Jan 2014 to Jan 2015. Overall, 235 patients with thalassemia major and 235 healthy controls were enrolled. Assessment of anti-Toxoplasma antibodies in sera samples was performed using commercial ELISA kits. In order to the molecular investigate of T. gondii in blood samples, a relatively new molecular assay, LAMP technique based on Toxoplasma SAG1 gene was conducted for the first time. The specificity of LAMP outer primers for the T. gondii detection was confirmed by sequencing the purified PCR product. Results: 51.9% of thalassemia patients and 34.8% of healthy controls were positive for anti-Toxoplasma IgG antibodies, which the difference was statistically significant (P<0.01). In terms of anti-Toxoplasma IgM antibody, 3.4% of thalassemia patients and 2.1% of healthy individuals were positive, which the difference was not statistically significant (P=1). Based on SAG1-LAMP, 9.78% of the thalassemia patients and 5.95% of healthy controls were positive for T. gondii DNA, which the difference was not statistically significant (P≤0.230). Conclusion: Thalassemia patients, probably due to repeated blood transfusion and consequently, immune deficiency, are at risk of transmitting Toxoplasma infection more than healthy people. Therefore, screening of Toxoplasma infection in blood transfusion centers may be effective in the prevention of toxoplasmosis in these patients.