We evaluated the relationship between plasma levels of anti-Müllerian hormone (AMH) and the number of antral follicles at the restart of the follicular wave in crossbred Holstein cows reared under extensive grazing systems over 2500 m above sea level. The study included 140 cows from 15 farms that were in average at the 75.3 ± 2.10 d post partum. Animals were synchronized according to the following regime: day 0 = intravaginal progesterone releasing device (IPD) + estradiol benzoate (EB); day 7: withdrawal of IPD + prostaglandin; and day 8: EB, for restart of the follicular wave on day 11. On this day 11, antral follicle counts (AFCs) were made by transrectal ultrasound, and a plasma sample was taken for the determination of AMH. The mean AMH plasma level was 0.06 ± 0.03 ng/ml and the mean AFC was 17.26 ± 0.38 follicles. A strong positive linear correlation was found between these two variables (r = 0.783, r = 0.613, P < 0.0001). Cows were categorized according to AMH concentration as high (>0.09 ng/ml), intermediate (0.09–0.05 ng/ml) or low (<0.05 ng/ml). Cows with high AMH presented a higher AFC (25.0 ± 2.21 follicles) than those with low AMH (14.08 ± 2.68 follicles; P < 0.001. Our results suggest that the cut-off value of AMH = 0.09 ng/ml may be useful for selecting donors in multiple ovulation embryo transfer programs involving cows with these characteristics. Our data further suggest that AMH plasma concentration correlates with AFC and can be used as an endocrine biomarker of the number of antral follicles present at a given moment of the estrous cycle in crossbred Holstein cows raised at altitudes above 2500 m.