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Subclinical ketosis (SCK) is defined as elevated concentrations of ketone bodies in the absence of clinical signs of ketosis. It is an important metabolic disease in dairy cattle during early lactation and is associated with losses in milk production and several other periparturient diseases. Limited information is available regarding the prevalence of SCK in dairy herds in Iran. The objectives of this study were (i) to determine the incidence of SCK in the dairy herds in Kerman province of Iran using serum β-hydroxybutyrate (BHB) concentrations, and (ii) to investigate the relationship between serum concentrations of BHB and glucose of cows with SCK. In the present study, 90 multiparous Holstein cows (4-6 years old) from 11 commercial dairy herds were evaluated 3-4 weeks after calving. The distribution of blood BHB concentrations seemed to suggest a cut-off point of 1200 μmol/L between cows with and without SCK. At this cut-off point, 14.4% of tested cows (13/90) were classified as subclinically ketotic, with the prevalence rate within herd ranging from 10% to 20%. Cows with SCK were detected in all the investigated dairies except one. Blood glucose concentrations in cows with SCK were significantly lower (p ≤ 0.05) than in cows without SCK, and serum BHB and glucose concentration were inversely correlated (r = −0.43, p ≤ 0.05). The results suggest that, using a cut-off of 1200 μmol/L, BHB concentrations can be used during early lactation for diagnosis and to make management decisions for prevention and treatment.