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This study aimed to conduct a comprehensive investigation to demonstrate early pregnancy detection using thermography in heifers and cows. A total of 60 heifers (n : 30) and cows (n : 30) were divided into two groups as pregnant (n : 15 heifers, n : 15 cows) and non-pregnant (n : 15 heifers, n : 15 cows) according to the day 28 of gestation. Thermographic images were taken from the vulvar and anal regions on alternate days from D0 to D20. Blood samples were collected to determine estrogen and progesterone concentrations. The mean temperature difference between the anal and vulvar regions (ΔT °C) was used in the statistical analyses. Based on the hormonal profiles, no abnormalities were observed for follicular waves or luteal profiles in heifers and cows. The ΔT °C values between heifers and cows and between days were statistically significant (P < 0.001). In thermographic analyses, the differences observed in other main effects and interactions of the group, sampling time, and pregnancy were not statistically significant (P > 0.05). In receiver operating characteristic (ROC) analyses, it was concluded that the ΔT °C value of ≤ 2.9 °C (100% Se - 61.9% Sp) was highly correlated with pregnancy diagnosis in cows on day six after artificial insemination (AI) (P < 0.001). In conclusion, it was determined that the clinical application of thermography can be used for the detection of pregnancy on day six after AI in cows. However, further studies are needed to determine heifers' thermographic characteristics and profiles. • Thermography is a useful tool for monitoring reproductive activity in cattle. • Pregnant cows can be detected by thermography on the 6th day after insemination. • The cows that have ΔT ≤ 2.9 °C on D6 can be determined as pregnant. • Pregnancy can not be determined by thermography in the first 20 days in heifers. [ABSTRACT FROM AUTHOR]