Fatty acids in milk are one of the most important components of milk. The aim of this study was to determinate relationships between groups of fatty acids and body condition score change in Czech Fleckvieh cows. Fatty acids were classification along its length of chain and its source of creation. To analysis was included total of 50 Czech Fleckvieh cows with different order of lactation. During the first 4 weeks of lactation, milk samples were collected at a weekly interval and body condition score was assessed. Statistical analyses were performed using Microsoft Office Excel and the procedures MEANS and GLM of SAS 9.1. During the first four week of lactation, the proportions of short- and medium-chain fatty acids as well as de novo synthesised fatty acids increased. Moreover the cows with a greater body condition score change mobilized storage depot fat more intensively, which resulted in higher proportions of dietary and depot fatty acids since week 1 of lactation, and long-chain fatty acids since week 2 of lactation. On the contrary, the animals with only a small body condition score change exhibited high proportions of short- and medium-fatty acids as well as de novo synthesised fatty acids in the most part of the period analysed. This indicates abut negative energy balance in early part of lactation and its compensation. The results also confirm the relationships between different groups of fatty acids, body condition score and negative energy balance. As more accurate for evaluation of intensity of negative energy balance was confirmed rather classification along source of creation. These results further emphasize importance the monitoring of body condition, milk composition and good herd management in first part of lactation.