Background Herein we assessed the sero-molecular prevalence of Toxoplasma infection in diabetic pregnant women, southwestern Iran. Methods Totally, 264 sera were collected from diabetic pregnant women for detection of specific IgM, IgG and IgG avidity. DNA from buffy coats were used for B1-nested PCR. Results In ELISA, out of 264 pregnant women with diabetes, 79 (29.92%) were showed positive. Of these, 75 (28.4%) were IgG +/ IgM _ and 4 (1.5%) were IgG +/IgM +. By IgG avidity, all cases with only IgG titers had high avidity, while 4 patients positive for both IgG and IgM had low avidity. Based on nested-PCR method, of 79 seropositive samples, 16 (20.25%) and among the 185 seronegative ones, 5 (2.7%) were positive. Molecular positive cases were most frequent (13.69%) in T1DM patients. Conclusion Remarkable positive cases were revealed in this sero-molecular study, implicating proper management particularly for those in early pregnancy.