The purpose of this study was to determine the relationship between non-esterified fatty acids (NEFA) at calving, body condition score (BCS), and milk yield in Holstein cows from central Chile. The study was conducted on two commercial dairy herds with an average milk yield of 8000 kg per lactation. Cows were fed a total mixed ration based on alfalfa hay, corn silage and concentrate. Blood samples were taken from 350 cows at calving and NEFA were determined. BCS at calving and at 60 days post partum, accumulated milk yield at 30, 60 and 100 days of lactation were determined. Data were analyzed using the mixed procedure of SAS (2003). Mean values for NEFA at calving, and BCS were 0.90 ± 0.47meq/l, and 3.6 ± 0.3, respectively. In a multivariable level model, considering BCS at calving and parity as independent variables, the relationship between NEFA at calving and milk yield at 100 days of lactation was quadratic, with lower milk yield at high and low values of NEFA and higher milk yield at intermediate levels of NEFA (R2= 0.27; p<0.001). A positive correlation was observed between BCS and NEFA at calving (r= 0.26; p<0.05). Concentration of NEFA increased when the change in BCS between calving and 60 days post calving was higher. It is concluded that NEFA at calving, was associated in a quadratic form with milk yield at 100 days of lactation.