A total of 302 serum samples collected from Chitawan (159) and Mustang (143) districts of Nepal were included in this study. Anti-toxoplasma antibody was detected using micro-latex agglutination (MLA) and ELISA methods. An overall positive rate was found to be 57.9%. The positive rate in Chitawan was significantly higher (64.1%) (less than 1,000 m altitude) compared to that in Mustang (51.0%) (more than 3,000 m altitude) (p < 0.05). Females in Chitawan showed significantly higher positive rate (71.2%) compared to males (56.9%) (p < 0.05). On the contrary, though insignificantly, males showed higher positive rate (57.9%) compared to that of females (43.3%) in Mustang. Almost equal positive rate was observed among males in both study area. Females in Chitawan showed significantly higher (71.2%) positive rate compared to their counterparts in Mustang (43.3%) (p < 0.001). A slight increase in positive rate with age was observed in Chitawan while in Mustang a decreasing trend was noticed. Ethnically though statistically not significant, Indo-Aryans showed a higher positive rate (69.2%) compared to the positive rate shown by Tibeto-Burmans (63.1%) in Chitawan while the reverse was true in Mustang (Tibeto-Burmans: 53.8% and Indo-Aryans: 38.4%). Interestingly, 2.9% and 1.3% of MLA positive samples showed toxoplasma IgM antibody. None of the IgM positive samples were positive for toxoplasmic antigens.