Objective: To evaluate the possible effect of methotrexate (MTX) on rat ovaries by measuring serum antimullerian hormone (AMH), the novel marker of the ovarian reserve. Methods: Pretreatment serum AMH levels were measured in 15 Wistar albino rats. MTX was given in 1 mg/kg dose in days 1, 3, 5, and 7. Serum AMH levels were measured twenty-four hours after each MTX administration. Pre- and post-treatment serum AMH levels were compared. Results: Pretreatment median serum AMH was 102.4 ng/mL (25%: 41.9; 75%: 179.8). The median serum AMH levels were 70.6 ng/mL (25%: 54.08; 75%: 125.5); 136.1 ng/mL (25%: 57.3; 75%: 223.09); 121.2 ng/mL (25%: 52.5; 75%: 151.5); and 104.7 ng/mL (25%: 65.8; 75%: 265.5) after the first, second, third, and fourth methotrexate administrations, respectively. The ratio of the final (eighth day) median serum AMH level to the pretreatment median AMH level was 1.27 (25%: 0.84 and 75%: 2.57). Wilcoxon related samples test showed that final AMH was significantly higher as compared to the second day AMH measurement (p = 0.041). Conclusion: MTX administration did not cause a statistically significant change between pretreatment and final serum AMH levels in rats. There was no decrease in AMH levels indicating a decrease in ovarian reserve.