Aim: The purpose of our study was to determine the content of trans fatty acids in early human breast milk as an indicator of dietary exposure in a sample of Roma breast-feeding women and in a sample of women from the general Czech population. Methods: We collected samples of early human milk from 43 Prague women from the general population and 21 Roma women. After lipid extraction, the fatty acids were converted into methyl esters (FAMEs). Finally, gas chromatography with flame ionization detector (GC-FID) analysis on a CP-Sil 88 column was used to determine C18:1 trans monoenic fatty acid levels and total trans isomers fatty acid levels in human milk. Results: A significantly higher content of C18:1 trans fatty acid isomers was detected in human milk fat from Roma mothers than in women of the general population (2.73 vs. 2.09%, p < 0.05). Both groups monitored did not differ in the representation of total fatty acid trans isomers. Differences in the frequency of consumption of certain TFA sources (butter, fried crisps) were established. Conclusions: The study proved a higher fatty acid trans isomers content in Roma breast-feeding mothers in the Czech Republic, and this is probably related to their bad eating habits.