The aim of this study was to evaluate the relationships between milk fatty acid group contents and subsequent reproductive performance in Holstein cows. A total of 27 cows in the 1st to 4th lactations were included in the evaluation. Cows were divided into primiparous and multiparous groups. Daily milk yields increased in the first 8 weeks of lactation. Body condition score decreased until the third month of lactation. As the cows gradually recovered from the negative energy balance, the contents of saturated fatty acids (SFAs) increased, whereas the contents of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) decreased. The contents of SFAs and MUFAs were significantly (P < 0.01-0.05) correlated (r = -0.279 to r = 0.275) with all the reproductive traits evaluated (calving to first insemination interval, days open, and insemination index). PUFA was only correlated (P < 0.01) with calving to first insemination interval (r = -0.141). High milk SFA contents were associated with improved values of days open and insemination index, whereas opposite tendencies were observed for MUFA. It was concluded that the relationship was confirmed between the changes of different milk fatty acid group contents in early lactation, used as negative energy balance indicators, and subsequent reproduction results.