Copper (Cu) and zinc (Zn) are trace minerals with multiple biological functions, playing roles in fetal development and immune regulation. Despite their known significance, research on Cu and Zn administration and supplementation for pregnant mares is insufficient. This study aims to evaluate Cu and Zn serum concentrations monthly throughout the year and through pregnancy in Polish Konik mares. The study was conducted in 2020 on 36 mares from the Polish state stud farm, with 24 mares in the pregnant group and 12 mares in the non-pregnant group. Monthly blood samples were collected, and serum Cu and Zn concentrations were measured. The total Cu concentration in the serum of all mares was higher (p < 0.0001) in August and September (13.98 ± 3.00 μmol/L) than in May, June, July, and November (11.04 ± 2.74 μmol/L). Similarly, the total Zn concentration was higher (p < 0.0001) in August, September, October, November, and December (19.80 ± 9.72 μmol/L) than in May, June, and July (14.50 ± 6.94 μmol/L). No evidence of a linear relation between Cu and Zn concentrations was demonstrated (slopes difference p < 0.001). No differences in Cu (p > 0.05) or Zn (p > 0.05) concentrations were found between pregnant (Cu: 12.08 ± 3.01 μmol/L; Zn: 18.02 ± 9.03 μmol/L) and non-pregnant (Cu: 11.23 ± 2.51 μmol/L; Zn: 18.59 ± 10.37 μmol/L) mares in any of the examined months of the year. In conclusion, the month of the year, rather than pregnancy, affects serum Cu and Zn concentrations.